Cartridges, kits, and methods for enhanced detection and quantification of analytes

ABSTRACT

Devices, systems, and methods for detecting molecules of interest within a collected sample are described herein. In certain embodiments, self-contained sample analysis systems are disclosed, which include a reusable reader component, a disposable cartridge component, and a disposable sample collection component. The reader component may communicate with a remote computing device for the digital transmission of test protocols and test results. In various disclosed embodiments, the systems, components, and methods are configured to identify the presence, absence, and/or quantity of particular nucleic acids, proteins, or other analytes of interest, for example, in order to test for the presence of one or more pathogens or contaminants in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 ofInternational Application No. PCT/US2016/042688, filed Jul. 16, 2016,which claims priority to U.S. Provisional Application No. 62/194,101,filed Jul. 17, 2015, the entire contents of each of which areincorporated herein by reference.

TECHNOLOGICAL FIELD

The present technology relates generally to the field of moleculedetection. In particular, the technology relates to microfluidicdevices, systems, and methods for detecting the presence, absence and/orquantity of one or more particular analytes within a collected sample.

BACKGROUND

Conventional technologies for identifying the presence, absence and/orquantity of nucleic acids, proteins, and/or other molecules of interestwithin a sample often require expensive laboratory equipment and theexpertise of highly-trained medical professionals. Consequently, suchanalyses are typically performed within laboratories or medicalfacilities. Such molecule detection can be important, for example, todetect the presence of pathogens, disease, contamination, overdoses, andpoisonings within a person or other animal or within the environment.Unfortunately, today, individuals may face long waits before the propertests can be performed and before the results can be generated andanalyzed. Due to the long waits and the inconvenience of traveling to alaboratory or medical facility, illnesses and contaminations oftenspread and may cause substantial harm before the presence of saidillness or contamination is even identified.

SUMMARY

There is a significant need for improved molecule detection andquantification technologies. Described herein are devices that maydetect molecules of interest in less time and with less technicalexpertise than the conventional devices used today. The devices may beutilized by consumers in non-clinical settings, for example, in schools,places of employment, and in the home. In addition, the devices may beused by consumers upon entering a pharmacy or healthcare facility, andmay generate results quickly so that results are available by the timethe consumer talks with a pharmacist or healthcare practitioner. Thedevices herein also may be configured to minimize biohazard risks.

One aspect of the disclosure is directed to a system for detectingmolecules. In various embodiments, the system includes a cartridgedevice, a reader device removably coupled to the cartridge device, and asample collection device.

Sample preparation reagents may be used in the system and may include aplurality of magnetic particles each having surface-bound affinitymolecules, a plurality of detector agents which may each include asignaling agent, a plurality of amplification reagents, and/or aplurality of agents to facilitate access to a target analyte and bindingbetween the target analyte and the surface-bound affinity molecules andthe detector agents.

In accordance with one aspect, a sample analysis cartridge is providedfor detecting at least one of a presence, absence, or quantity of one ormore analytes. The sample analysis cartridge may include an inputtunnel, a reservoir, a reagent shuttle, and/or a sensor, each of whichmay be within a housing of the cartridge. The input tunnel may extendfrom an aperture and may be configured to permit insertion of a samplecollection device having a distal portion adapted to be exposed to asample. The reservoir may be configured to hold a fluid, which may ormay not be pre-filled within the reservoir. The reagent shuttle may bedisposed between the reservoir and the aperture in a first position andthe reagent shuttle may have a first end and a second end. The reagentshuttle may be configured to house a reagent ball comprising reagents(e.g., sample preparation reagents) between the first and second ends.The first end may be configured to seal the reservoir from the inputtunnel in the first position. The reagent shuttle may be designed tomove within the input tunnel when subjected to a force greater than athreshold force to a second position such that the reagent ball and thesample move into the reservoir. The reservoir may be continuously sealedfrom the input tunnel proximal to the reagent shuttle during movementfrom the first position to the second position. The sensor may beconfigured to analyze the fluid mixed with the reagent ball and thesample and further configured to generate a signal indicative of atleast one of the presence, absence, or quantity of the one or moreanalytes within the sample.

The second end of the shuttle may have an opening sized to wipe excesssample from a tip of the sample collection device such that, at most, apredetermined volume of the sample is mixed in the fluid within thereservoir. The reservoir may be sealed via the sample collection devicepartially inserted within the second end of the shuttle during movementfrom the first position to the second position. The cartridge may haveone or more locking members configured to irreversibly lock the samplecollection device within the input tunnel in the second position.

The shuttle may include one or more sample compartments configured tohouse, at most, a predetermined volume of the sample and one or morereagent ball compartments configured to house the reagent ball and,optionally, additional reagent balls. In some embodiments, the one ormore sample compartments and the one or more reagent ball compartmentsare not exposed to the fluid within the reservoir in the first position.The one or more sample compartments and the one or more reagent ballcompartments may be exposed to the fluid within the reservoir in thesecond position. The shuttle may have a compartment divider configuredto divide at least one sample compartment from at least one reagent ballcompartment. The compartment divider may have a slot configured tofacilitate mixing when disposed within the sample preparation reservoirin the second position.

The reagents may include one or more of a plurality of solid particles,a plurality of affinity molecules, and/or a plurality of signalingagents. The reagents may include a plurality of magnetic particlesconfigured to be magnetically held over a working electrode of thesensor. At least one magnetic particle of the plurality of magneticparticles may be configured to be indirectly bound to a signaling agent.

The cartridge may include an analysis channel, e.g., within thecartridge housing. At least a portion of the sensor may be disposed inthe analysis channel and the fluid mixed with the reagent ball and thesample may travel to at least the portion of the sensor via the analysischannel.

The cartridge may include a contact switch, a sealing materialconfigured to fluidicly seal the fluid within the reservoir, and/or aseal piercer and each of those components may be within the cartridgehousing. Insertion of the sample collection device within the inputtunnel may cause: (i) the shuttle to move from the first position to thesecond position, (ii) the seal piercer to pierce the sealing material tovent the fluid in the reservoir, and/or (iii) activation of the contactswitch.

In accordance with another aspect, a sample analysis cartridge isprovided for detecting at least one of a presence, absence, or quantityof one or more analytes. The sample analysis cartridge may include aninput tunnel, a reservoir, a sealing material, and/or a seal piercer,each of which may be within a housing of the cartridge. The input tunnelmay extend from an aperture and the input tunnel may be configured topermit insertion of a sample collection device having a distal portionadapted to be exposed to a sample. The reservoir may be configured tohold a fluid, which may or may not be pre-filled. The sealing materialmay be configured to fluidicly seal the fluid within the reservoir. Theseal piercer may be disposed partially within the input tunnel and theseal piercer may be configured to be contacted by the sample collectiondevice within the input tunnel and to move, responsive to force appliedby sample collection device, to cause the sealing material to be piercedto vent the fluid in the reservoir.

The seal piercer may be configured to move in a first direction and asecond direction, different from the first direction, to pierce thesealing material. The first direction may be substantially parallel tomovement of the sample collection device within the input tunnel and thesecond direction may be substantially perpendicular to the firstdirection. The seal piercer may include one or more piercers. The sealpiercer may include a slider configured to move in a first direction andthe one or more piercers may be configured to move in a seconddirection, different from the first direction, to pierce the sealingmaterial.

The reservoir may be a sample preparation reservoir and may beconfigured to hold sample preparation reagents which may be within thefluid and/or introduced, e.g., via introduction of a reagent ball(s).The cartridge may also include a wash reservoir and/or a substratereservoir. The seal piercer may be configured to cause the sealingmaterial to be pierced to vent respective fluids in the samplepreparation reservoir, the wash reservoir, and the substrate reservoir.The seal piercer may include an engager disposed within the input tunneland the engager may be configured to engage an engagement zone of thesample collection device when the sample collection device is within theinput tunnel.

The cartridge may include a contact switch which may be disposed on acircuit board within the housing. The contact switch may be configuredto be activated upon insertion of the sample collection device withinthe input tunnel. Movement of the seal piercer may cause activation ofthe contact switch. The seal piercer may be configured to sequentiallypierce the sealing material over the sample collection reservoir, thewash reservoir, and the substrate reservoir, in any order. The sealpiercer may be configured to move out of one or more holes pierced inthe sealing material after piercing to vent the fluid in the reservoir.

The cartridge may include a contact switch and a shuttle disposedbetween the reservoir and the aperture in a first position. The shuttlemay have a first end and a second end and the first end may beconfigured to seal the reservoir from the input tunnel in the firstposition. The reagent shuttle may be configured to move within the inputtunnel to a second position wherein the sample is moved into thereservoir. Insertion of the sample collection device within the inputtunnel may cause: (i) the shuttle to move from the first position to thesecond position, (ii) the seal piercer to pierce the sealing material tovent the fluid in the reservoir, and/or (iii) activation of the contactswitch. The cartridge may include one or more locking members configuredto irreversibly lock the sample collection device within the inputtunnel in the second position. The one or more locking members mayirreversibly lock the sample collection device within the input tunnelduring partial and/or full insertion of the sample collection devicewithin the input tunnel.

Insertion of the sample collection device within the input tunnel maycause the seal piercer to pierce the sealing material to vent the fluidin the reservoir before the shuttle moves from the first position to thesecond position. Alternatively, or additionally, insertion of the samplecollection device within the input tunnel may cause the seal piercer topierce the sealing material to vent the fluid in the reservoir duringmovement of the shuttle from the first position to the second position.

In accordance with yet another aspect, a sample analysis cartridge isprovided for detecting at least one of a presence, absence, or quantityof one or more analytes. The sample analysis cartridge may include aninput tunnel, a reservoir, an analysis channel, and/or a circuit board,each of which may be within a housing of the cartridge. The input tunnelmay extend from an aperture and the input tunnel may be configured topermit insertion of a sample collection device having a distal portionadapted to be exposed to a sample. The reservoir may be configured tohold a fluid and may be configured to receive the sample on the distalportion of the sample collection device. The analysis channel may beconfigured to receive, from the reservoir, the fluid having the sampleand reagents comprising a plurality of magnetic particles mixed therein.The circuit board may include a sensor having a working electrode andthe sensor may be configured to be exposed to the mixed fluid in theanalysis channel and to generate a signal indicative of at least one ofthe presence, absence, or quantity of the one or more analytes withinthe sample. The working electrode may be masked with a plurality ofstriations configured to promote homogenous distribution of theplurality of magnetic particles over the working electrode and topromote resistance to movement of the plurality of magnetic particlesoff of the working electrode.

In accordance with yet another aspect, a kit is provided for detectingat least one of a presence, absence, or quantity of one or moreanalytes. The kit may include a sample collection device, a sampleanalysis cartridge, and/or a sample analysis reader. The samplecollection device may have a distal portion adapted to be exposed to asample. The sample analysis cartridge may include an input tunnel, areservoir, an analysis channel, and/or a circuit board, each of whichmay be within a housing of the cartridge. The input tunnel may extendfrom an aperture and may be configured to permit insertion of the samplecollection device. The reservoir may be configured to hold a fluid andconfigured to receive the sample on the distal portion of the samplecollection device. The analysis channel may be configured to receive,from the reservoir, the fluid having the sample and reagents comprisinga plurality of magnetic particles mixed therein. The circuit board mayinclude a sensor configured to be exposed to the mixed fluid in theanalysis channel and to generate a signal indicative of at least one ofthe presence, absence, or quantity of the one or more analytes withinthe sample. The sample analysis reader may be configured to receive thesample analysis cartridge. The sample analysis reader may have first andsecond magnetic generators configured to be disposed adjacent to asingle working electrode of the sensor when the sample analysiscartridge is inserted in the sample analysis reader. The first andsecond magnetic generators may be configured to generate a magneticfield over the length of the single working electrode to promotehomogenous distribution of the plurality of magnetic particles over thelength of the single working electrode.

Receipt of the sample analysis cartridge by the sample analysis readermay cause electric coupling between the sample analysis cartridge andthe sample analysis reader. The sample analysis cartridge may beconfigured to transmit the signal indicative of at least one of thepresence, absence, or quantity of the one or more analytes within thesample to the sample analysis reader for processing. The sample analysisreader may be configured to transmit the processed signal indicative ofat least one of the presence, absence, or quantity of the one or moreanalytes within the sample to a computer. The kit may include a computerreadable medium with instructions that, when executed by a processor ofthe computer, cause a display of the computer to display informationindicative of the presence, absence, and/or quantity of one or moretarget analytes.

A housing of the sample analysis cartridge may include a bottom surfacehaving a magnetic generator depression. Receipt of the sample analysiscartridge in sample analysis reader may cause the first and secondmagnetic generators to move partially within the magnetic generatordepression. The magnetic generator depression may be disposed adjacentthe single working electrode.

In accordance with another aspect, a sample analysis cartridge isprovided for detecting at least one of a presence, absence, or quantityof one or more analytes. The sample analysis cartridge may include aninput tunnel, a reservoir, a heater, an analysis channel, and/or asensor, each of which may be within a housing of the cartridge. Theinput tunnel may extend from an aperture and the input tunnel may beconfigured to permit insertion of a sample collection device having adistal portion adapted to be exposed to a sample. The reservoir may beconfigured to hold a fluid and configured to receive the sample on thedistal portion of the sample collection device. The reservoir mayinclude an outlet having a phase-changeable material therein to occludean entire cross-section of the outlet. The heater may be configured toheat the phase-changeable material such that the phase-changeablematerial does not occlude the entire cross-section of the outlet. Theanalysis channel may be configured to receive, from the reservoirthrough the outlet, the fluid having the sample and reagents comprisinga plurality of magnetic particles mixed therein. The sensor may beconfigured to be exposed to the mixed fluid in the analysis channel andto generate a signal indicative of at least one of the presence,absence, or quantity of the one or more analytes within the sample. Theheater may be masked with a masking material configured to electricallyisolate the heater from the sensor. The masking material may be a soldermask.

The cartridge may include a wash reservoir and a wash reservoir heater.The wash reservoir may be configured to hold a wash fluid and mayinclude a wash reservoir outlet having a phase-changeable materialtherein to occlude an entire cross-section of the wash reservoir outlet.The wash reservoir heater may be configured to heat the phase-changeablematerial in the wash reservoir outlet such that the phase-changeablematerial does not occlude the entire cross-section of the wash reservoiroutlet so that the wash fluid enters the analysis channel and travels tothe sensor. The wash reservoir heater may be masked with a maskingmaterial configured to electrically isolate the wash reservoir heaterfrom the sensor.

The cartridge may include a substrate reservoir and a substratereservoir heater. The substrate reservoir may be configured to hold asubstrate fluid and may include a substrate reservoir outlet having aphase-changeable material therein to occlude an entire cross-section ofthe substrate reservoir outlet. The substrate reservoir heater may beconfigured to heat the phase-changeable material in the substratereservoir outlet such that the phase-changeable material does notocclude the entire cross-section of the substrate reservoir outlet sothat the substrate fluid enters the analysis channel and travels to thesensor. The substrate reservoir heater may be masked with a maskingmaterial configured to electrically isolate the substrate reservoirheater from the sensor.

In accordance with yet another aspect, a sample analysis cartridge isprovided for detecting at least one of a presence, absence, or quantityof one or more analytes. The sample analysis cartridge may include asample preparation reservoir, a substrate reservoir, an analysischannel, a fluidic isolator, and/or one or more heaters, each of whichmay be within a housing of the cartridge. The sample preparationreservoir may be configured to hold a fluid and may be configured toreceive a sample from a sample collection device. The sample preparationreservoir may include a sample preparation reservoir outlet havingphase-changeable material therein to seal the sample preparationreservoir outlet. The substrate reservoir may be configured to hold afluid comprising a chemical substrate. The substrate reservoir mayinclude a substrate reservoir outlet having phase-changeable materialtherein to seal the substrate reservoir outlet. Each of the samplepreparation and substrate reservoirs may be, at least at times, in fluidcommunication with the analysis channel. The fluidic isolator may bephase-changeable material. At least one of the one or more heaters maybe configured to heat phase-changeable material within the samplepreparation reservoir outlet such that the phase-changeable materialunseals the sample preparation reservoir outlet to permit the fluidhaving the sample mixed therein to flow into the analysis channel. Atleast one of the one or more heaters may be configured to heatphase-changeable material of the fluidic isolator after unsealing thesample preparation reservoir outlet such that phase-changeable materialof the fluidic isolator flows into the analysis channel to fluidiclyisolate the sample preparation reservoir from the substrate reservoir.At least one of the one or more heaters may be configured to heatphase-changeable material within the substrate reservoir outlet, afterthe fluidic isolator fluidicly isolates the sample preparation reservoirfrom the substrate reservoir, such that the phase-changeable materialunseals the substrate reservoir outlet to permit the fluid comprisingthe chemical substrate to flow into the analysis channel, but not intothe sample preparation reservoir.

The one or more heaters may include a sample preparation reservoirheater, a fluidic isolator heater, and/or a substrate reservoir heater.The sample preparation reservoir heater may be configured to heat thephase-changeable material within the sample preparation reservoiroutlet. The fluidic isolator heater may be configured to heat thephase-changeable material of the fluidic isolator. The substratereservoir heater may be configured to heat the phase-changeable materialwithin the substrate reservoir outlet. The sample preparation reservoirheater, the fluidic isolator heater, and the substrate reservoir heatermay each be masked with a masking material configured to electricallyisolate the respective heater from a sensor in the analysis channel.

The cartridge may include a wash reservoir configured to hold a washfluid. The wash reservoir may include a wash reservoir outlet havingphase-changeable material therein to seal the wash reservoir outlet. Atleast one of the one or more heaters may be configured to heatphase-changeable material within the wash reservoir outlet, after thefluidic isolator fluidicly isolates the sample preparation reservoirfrom the substrate reservoir, but before the one or more heaters heatsthe phase-changeable material within the substrate reservoir outlet,such that the phase-changeable material unseals the wash reservoiroutlet to permit the wash fluid to flow into the analysis channel towash signaling agents unbound to magnetic particles from the samplepreparation reservoir off a sensor in the analysis channel. The fluidhaving the chemical substrate may be configured to wash signaling agentsunbound to magnetic particles from the sample preparation reservoir offa sensor in the analysis channel.

In accordance with another aspect, a sample analysis cartridge isprovided for detecting at least one of a presence, absence, or quantityof one or more analytes. The sample analysis cartridge may include aninput tunnel, a reservoir, a shuttle, and/or a sensor, each of which maybe within a housing of the cartridge. The input tunnel may extend froman aperture and the input tunnel may be configured to permit insertionof a sample collection device having a distal portion adapted to beexposed to a sample fluid. The reservoir may be configured to hold afluid, which may or may not be pre-filled. The shuttle may be disposedbetween the reservoir and the aperture in a first position. The shuttlemay have a first end and a second end and may define a samplecompartment between the first and second ends. The sample compartmentmay be configured to receive the sample fluid compressed from the distalportion of the sample collection device. The shuttle may be configuredto move within the input tunnel when subjected to a force greater than athreshold force to a second position such that the sample compartmenthaving the sample fluid is moved into the reservoir. The sensor may beconfigured to be exposed to the fluid mixed with the sample fluid andthe sensor may be further configured to generate a signal indicative ofat least one of the presence, absence, or quantity of the one or moreanalytes within the sample fluid.

The shuttle may further define a reagent ball compartment between thefirst and second ends. The reagent ball compartment may be configured tohouse one or more reagent balls comprising reagents. The reagent ballcompartment may be outside the reservoir in the first position and inthe reservoir in the second position. The sample compartment may beconfigured to receive, at most, a predetermined volume of the samplefluid compressed from the distal portion of the sample collectiondevice. The cartridge may include an overflow compartment configured toreceive a volume of the sample fluid from the sample compartment abovethe predetermined volume.

The reagent ball comprises reagents necessary to carry out amplificationof the target analyte. The reagent ball can be of any appropriate andshape, non-limiting examples of such include a diameter of between about1 mm to about 7 mm, or alternatively from about 2 mm to about 5 mm, oralternatively about 3 mm, or alternatively less than about 7 mm, oralternatively less than about 5 mm, or alternatively less than about 4mm. The reagent ball can be of any appropriate shape, e.g., spherical,cylindrical, conical or ellipsoid.

The components of the reagent ball are preselected for the analyte andits method for amplification and subsequent detection and/orquantification. In one aspect, the components of the reagent ballcomprise reagents for the detection or quantification of a hormone,other small molecule or a protein or fragment. In another aspect, thecomponents of the reagent ball comprise reagents for the detectionand/or quantification of a nucleic acid by a method that comprisesamplifying the nucleic acid.

A kit may be provided including the sample analysis cartridge and thesample collection device. The sample collection device may include awicking portion at the distal portion. The wicking portion may beconfigured to wick and absorb the sample fluid. The wicking portion maybe compressed to expel the sample fluid into the sample compartment. Thesample compartment may be configured to receive, at most, apredetermined volume of the sample fluid compressed from the distalportion of the sample collection device. The sample analysis cartridgemay further include an overflow compartment configured to receive avolume of the sample fluid from the sample compartment above thepredetermined volume. The wicking portion of the sample collectiondevice may be configured to wick and absorb sample fluid above thepredetermined volume to permit a user to meter a quantity of samplefluid compressed into the sample compartment and the overflowcompartment. At least some of the wicking portion may be slidablydisposed within a shroud of the sample collection device.

The sample analysis cartridge may further include one or more lockingmembers configured to irreversibly lock the sample collection devicewithin the input tunnel when the sample collection device is fullyinserted in the input tunnel. The sample collection device may furtherinclude a sample collection indicator configured to visually alert acollector based on a volume of sample fluid that has been collected. Thesample collection indicator may be a colored thread embedded in thewicking portion that becomes increasingly visually exposed as the volumeof sample collected increases.

In accordance with another aspect, compositions and method are providedfor detecting and/or quantifying at least one of a presence, absence, orquantity of a target analyte within a sample in a cartridge. The methodmay include mixing, within a fluid in a reservoir of the cartridge, aplurality of affinity molecules, a plurality of de-binding agents, aplurality of signaling agents, a plurality of competitor moleculespre-bound to competitor binding molecules, each of the plurality ofcompetitor molecules possessing a label, and the sample having aplurality of sample target analytes pre-bound to sample bindingmolecules; de-binding at least one competitor molecule from thepre-bound competitor binding molecule using at least one de-bindingagent of the plurality of de-binding agents; de-binding at least onesample target analyte from the pre-bound sample binding molecule usingat least one de-binding agent of the plurality of de-binding agents;binding the label of the de-bound competitor molecule to a signalingagent of the plurality of signaling agents; binding the de-boundcompetitor molecule to an affinity molecule of the plurality of affinitymolecules; and/or generating a signal indicative of at least one of apresence, absence, or quantity of the sample target analyte within thecartridge. The reagent ball can be of many sizes, non-limiting examplesof such include having an diameter of between about 1 mm to about 7 mm,or alternatively from about 2 mm to about 5 mm, or alternatively about 3mm, or alternatively less than about 7 mm, or alternatively less thanabout 5 mm, or alternatively less than about 4 mm. While reagent ball isillustrated as a sphere, the disclosure is not limited thereto and manyshapes may be used and multiple reagent balls each containing the sameor different reagents also may be used.

In accordance with another aspect, compositions and methods are providedfor amplifying and detecting and/or quantifying at least one of apresence, absence, or quantity of a target analyte, e.g., a targetnucleic acid (deoxyribonucleic acid (DNA) or ribonucleic acid (RNA))within a sample in a cartridge. The method comprises or alternativelyconsists essentially of preparing a plurality of amplicons comprising aplurality of capture elements, by mixing within a fluid in a reservoirof the cartridge: a plurality of enzymes to facilitate an amplificationreaction, e.g., polymerases; reverse transcriptases; a plurality ofmagnetic beads having coupled thereto an affinity molecules; and aplurality of forward and reverse primers, that may or may not belabeled.

In a further aspect, provided herein are compositions and methods forthe use, wherein the reagent ball further contains a plurality offorward primers selected to amplify the target nucleic acid and havingcoupled to each thereto a spacer element and a capture element; aplurality of reverse primers selected to amplify the target nucleic acidand having coupled to each thereto a spacer element and a signalingagent or a signaling capture element; a plurality of nucleotides oranalogs thereof (dNTPs) for the amplification reaction.

In a further aspect, the reagent ball further contains a reverse primerselected to bind a target nucleic acid, the reverse primer comprising aspacer element that is directly or indirectly conjugated to a reportercapture element. In a further aspect, the reagent ball further containsan effective amount of reverse transcriptase, effective to facilitatethe amplification reaction.

In a yet further aspect, the reagent ball also contains a plurality ofreporter affinity element conjugated to a reporter element.

In a further aspect, the reagent ball also contains single strandedbinding proteins, e.g., from about 9 to about 18 amino acids, known tothose of ordinary skill in the art such as but not limited to SSB fromE. coli or GP 32 from phages.

In one aspect, the reagent ball further contains, a plurality of DNAtemplate control nucleic acids. In another aspect, the reagent ballalternatively or also contains a plurality of RNA template controlnucleic acids.

In a further aspect, the reagent ball may contain a plurality of reversetranscriptase-specific primers to facilitate reverse transcription ofRNA to cDNA. In a yet further aspect, the reagent ball may also containa plurality of reverse primers selected to serve as an internal controlhaving coupled to each thereto a spacer element and a signaling agent;and a plurality of forward primers selected to serve as an internalcontrol having coupled to each thereto a spacer element and a captureelement.

In addition to the labeled primers, the reagent ball can contain aneffective amount of a plurality of unlabelled primers designed toamplify at least the same target region but optionally flankingsequences to the target sequence. The presence of unlabeled primers canmake amplification more efficient since labelled primers may be moresterically hindered as they bind to other elements e.g. solid particlesor signaling agents.

The reagent ball can further contain an effective amount of one or lysisagents to free the target nucleic acid, templates and/or control(s) froma cell, microbe, or virus in the sample.

In a further aspect, the reagent ball contains an effective amount of ahelicase to unwind dsDNA for loading primers or RecA or its analoguessuch as UvsX or RAD51. Further, the reagent ball may contain mutL,RecFOR enzymes, UvsY.

In one aspect the amplification reagents are selected for any one ormore other amplification reactions, e.g., PCR methods or isothermalamplification. The reporter element and/or the capture element islocated at the 5′ termini of the nucleic acid or along the nucleic acidsequence, i.e., internal to the 5′ end. The reporter and/or the captureelement is covalently or non-covalently attached to the nucleic acid.

In one aspect the elements are provided in a sample reagent ball andmixed with the sample in the reservoir. Upon disintegration the reagentscome in contact with the target nucleic acid and hybridization of theprimers to the target nucleic acid and amplification of the targetthrough a series of enzymatically driven melting and reconstruction ofthe DNA or in the case of target RNA, a complementary DNA (cDNA)molecule is first created from the RNA target nucleic acid and thedouble stranded cDNA containing the target sequence then serves as thetemplate for further amplification. The reagent ball can be of manysizes, non-limiting examples of such include having an diameter ofbetween about 1 mm to about 7 mm, or alternatively from about 2 mm toabout 5 mm, or alternatively about 3 mm, or alternatively less thanabout 7 mm, or alternatively less than about 5 mm, or alternatively lessthan about 4 mm. While reagent ball is illustrated as a sphere, thedisclosure is not limited thereto and many shapes may be used andmultiple reagent balls each containing the same or different reagentsalso may be used. The spacer element separating the primer from a labelcomprises a polymer e.g., hexaethylene glycol or triethlyene glycol.Alternatively it can be a linear carbon polymer, e.g., hexane, pentane,containing from about 1 to about 18 carbon atoms or more.

As is apparent to the skilled artisan, combinations of the aboveembodiments, necessary to promote the specific amplification of a targetnucleic acid, are intended within the scope of this disclosure.

The reagents and amounts thereof are pre-selected to facilitate thespecific amplification of the target nucleic acids. For the purpose ofillustration, non-limiting examples of reverse transcriptases aremoloney murine leukemia virus (MMLV) or a derivative thereof or avianmyeoblastosis virus (AMV) or a derivative thereof. The preferred reversetranscriptase will be dependent on the target and may not be the samebetween pellets for different targets, as it can be appreciated that thereagent ball can be used in a variety of different testing applicationsby modifying primers sequences and capture elements, primerconcentrations, particle concentrations, affinity agents, lysis agents,polymerases, reverse transcriptases, and other enzymes to best match theoptimal condition for each type of target (e.g. HIV quantification vs.influenza detection may have different reaction conditions).

Polymerases can include several different types, within the stranddisplacement polymerase category, options include for instance Bsu DNApolymerase or a fragment thereof such as Bsu DNA polymerase largefragment, Bst DNA polymerase, or a fragment thereof such as Bst DNApolymerase large fragment, phi29 DNA polymerase. It can be appreciatedthat for polymerases and reverse transcriptases it is often a desiredproperty to have certain mutants such as polymerases lacking exonucleaseactivity or for the reverse transcriptase lacking RNase H activity.

Preferred reaction temperatures for an isothermal reaction can depend onthe method and the reaction conditions and can include around 65 degreesCelsius as is often the case for LAMP and 55 degrees Celsius for NickingEnzyme Amplification Reaction. A preferred but not limiting reactiontemperature range is between about 37 and 42 degrees for the reversetranscriptase portion of an amplification reaction if the target nucleicacid is an RNA. Helicase Dependent Amplification, Strand DisplacementAmplification, Recombinase Polymerase Amplification can all occur atabout 37 degrees Celsius or between 37 degrees Celsius and 42 degreesCelsius. FIG. 20 shows the temperature profile of an isothermal reactionoccurring in a reservoir at around 40 degrees Celsius.

It may be desired to include single-stranded binding proteins (SSB) tofacilitate several isothermal amplification techniques as these proteinshelp stabilize the unwinding and strand displacement polymerization ofcomplement strands during amplification. Examples include but are notlimited to RB 49 GP 32, RB 69 GP 32, T4 GP32, E. coli's SSB protein, andothers.

In some aspects, the reagent ball and the method further uses a helicaseto unwind dsDNA for loading primers. Non-limiting examples includeenzymes such as uvrD helicase from E. coli, T4 Gene 41 helicase, andmany others. Recombinases that facilitate primer loading into dsDNA forenzymatic melting of duplex DNA for primer annealing can include RecAfrom E. coli, RAD51 human recombinase, DMC1 human meiotic recombinase,or analogues from phage such as T4 UvsX, RB 49 UvsX, RB 69 UvsX and manyothers. As is known to those of ordinary skilled in the art,combinations of helicase and SSB are helpful for facilitating isothermalamplification. Accessory factors such as MutL can be added to facilitatehelicase dependent amplification. Combinations of recombinases and SSBcan be helpful in RPA and sometimes accessory factors such as RecFORfrom E. coli and/or UvsY from various phages are employed as well tofacilitate the reaction by helping the primary enzyme (RecA) or (uvrD)within recombinase polymerase amplification and helicase dependentamplification respectively. As appreciated by the skilled artisan, thereagent ball and/or reservoir can further contain any one or more of theabove reagents as necessary to facilitate the specific amplification ofthe target nucleic acid.

Primer concentrations for isothermal amplification reactions such asSDA, HDA, and RPA can be between 0.01 and 10 micromolar, preferablycloser to 0.5 micromolar. A LAMP Primer mix can be prepared with all 4or 6 (with Loop) primers. A 10× Primer Mix could contain: 16 μM FIP, 16μM BIP, 2 μM F3, 2 μM BE, 4 μM LoopF, 4 μM LoopB. dNTPs can be providedin concentrations such as between 1 micromolar and 500 micromolar,preferably around 200 micromolar. SDA, HDA, RPA may require a highamount of ATP as some of the enzymes that allow for enzymatic meltingand loading of primers into dsDNA require ATP to function and thereforeas much as 100 micromolar to 4 millimolar of ATP can be used within areaction.

Polymerase amounts can vary depending on the target but can be in therange of 1 unit to 1000 units per reaction. Reverse trancriptases canalso be provided at such a range for a successful reaction. Magnesium isan essential co-factor for polymerase activity and can be provided inthe reagent ball or in the reservoir at amounts well known in the artsuch as 5-50 millimolar, typically around 10 millimolar.

Methods and compositions for the non-covalent linkage of molecules thatalso can provide a label or signal for detection are known in the art.Non-limiting examples of such include avidin or streptavidin-biotinconjugation. Modifications to biotin are known in the art and intendedwithin the scope of this disclosure. Non-limiting examples of suchinclude biotin dT, biotin-TEG, dual biotin, PC biotin anddesthioBiotin-TEG commercially available from Integrated DNATechnologies (seeidtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/09/20/which-biotin-modification-to-use-,last accessed Jul. 16, 2016.) This disclosure also includes the use ofadditional conjugation chemistries for the linking of nucleic acids toproteins such as when a primer is conjugated directly to a signalingagent wherein in one aspect, the signaling agent is an enzyme such asHRP. Non-limiting examples of covalently joining a protein orpolypeptide to another moiety include linking the moiety to acrosslinking reactive group, e.g., carbodiimides, imidoesters, andmaleimides. See e.g., Bioconjugate Techniques, 3^(rd) Ed, Hermanson, G.T. (2013).

As is apparent to the skilled artisan, the components of the reagentball are selected to facilitate the amplification of the target nucleicacid and/or targets, and/or controls by the appropriate method. In oneaspect, the reagents are selected for rolling circle amplification(RCA), or loop-mediated isothermal amplification. In another aspect theyare selected for amplification by the (LAMP) method. In another aspectthey are selected for, strand displacement amplification (SDA). Inanother aspect they are selected for recombinase polymeraseamplification (RPA). In another aspect they are selected for, helicasedependent amplification (HDA). In another aspect they are selected for,polymerase spiral reaction (PSR). In another aspect they are selectedfor nicking enzyme amplification reaction (NEAR). As indicated above,each particular reaction type has its own preferred combination ofenzymes and components that allow efficient and selective amplificationof the target and/or targets, and/or internal control nucleic acids andare known to the skilled artisan.

Each of the affinity molecules of the plurality of affinity moleculesmay be bound to a solid particle. The solid particle may be formed ofmagnetically responsive material and/or may be formed ofnon-magnetically responsive material. The non-magnetically responsivematerial may be gold nanoparticles.

The plurality of sample target analytes pre-bound to sample bindingmolecules may include 25-hydroxy Vitamin D2 or 25-hydroxy Vitamin D3molecules pre-bound to vitamin D binding protein molecules. Theplurality of competitor molecules pre-bound to sample binding moleculesmay include 25-hydroxy Vitamin D2 or 25-hydroxy Vitamin D3 moleculeslabeled with biotin and prebound to vitamin D binding protein molecules.At least one of the plurality of affinity molecules, the plurality ofde-binding agents, the plurality of signaling agents, and the pluralityof competitor molecules pre-bound to competitor binding molecules may bestored within a reagent ball.

As noted above, the primers (forward and reverse for the target nucleicacid and control templates) are designed based on the nucleotidesequence of the target nucleic acid to be amplified and detected ifpresent. Methods to design optimal primers based on a target sequenceare known in the art, see e.g., simgene.com/Primer3; quill.com;molbiol-tools.caPCR; and ncbi.nlm.nih.gov/tools/primer-blast/, each lastaccessed on Jul. 15, 2016, and vary with the amplification methodutilized, e.g., rolling circle amplification (RCA), loop-mediatedisothermal amplification (LAMP), strand displacement amplification(SDA), recombinase polymerase amplification (RPA), helicase dependentamplification (HDA), polymerase spiral reaction (PSR), and nickingenzyme amplification reaction (NEAR).

In accordance with yet another aspect, a sample analysis cartridge isprovided for detecting at least one of a presence, absence, or quantityof one or more analytes. The sample analysis cartridge may include areagent ball, a reservoir, and/or a sensor, each of which may be withina housing of the cartridge. The reagent ball may include a plurality ofcompetitor molecules pre-bound to competitor binding molecules, each ofthe plurality of competitor molecules may possess a label or bound to asignaling agent. In another aspect, the reagent ball comprises, oralternatively consists essentially of, the reagents necessary foramplification and detection of a target nucleic acid. The reservoir maybe configured to hold a reservoir fluid, which may or may not bepre-filled in the reservoir. The reservoir may be configured to permitmixing of, within the reservoir fluid, a plurality of affinitymolecules, a plurality of de-binding agents, a plurality of signalingagents, the plurality of competitor molecules pre-bound to competitorbinding molecules, and a sample from a sample collection device, thesample having a plurality of sample target analytes pre-bound to samplebinding molecules. A de-binding agent of the plurality of de-bindingagents may be configured to de-bind a competitor molecule from thepre-bound competitor binding molecule or a sample target analyte fromthe pre-bound sample binding molecule. The label of the de-boundcompetitor molecule may be configured to bind to a signaling agent andthe de-bound competitor molecule may be configured to bind to anaffinity molecule of the plurality of affinity molecules. In anotheraspect, the reservoir is configured to hold a reservoir fluid, which mayor may not be prefilled in the reservoir. The reservoir configured topermit mixing of, within the reservoir fluid, a plurality of enzymes tofacilitate an amplification reaction, e.g., polymerases, reversetranscriptases; a plurality of magnetic beads having coupled thereto anaffinity molecule; a plurality of forward primers selected to amplifythe target nucleic acid and having coupled to each thereto a spacerelement; a plurality of reverse primers selected to amplify the targetnucleic acid and having coupled to each thereto a spacer element and asignaling agent or a signaling capture element; a plurality ofnucleotides or analogs thereof (dNTPs) for the amplification reaction; aplurality of DNA template control nucleic acids; a plurality of reverseprimers selected to serve as an internal control having coupled to eachthereto a spacer element and a signaling agent; and a plurality offorward primers selected to serve as an internal control having coupledto each thereto a spacer element and a capture element. In one aspectthe amplification reagents are selected for any one or more other PCRmethods or isothermal amplification.

In a further aspect, the reagent ball and/or the reservoir contains aneffective amount of a lysing agent to lyse a sample comprising a cell,e.g., a bacterial sample to release intracellular DNA, RNA and/orproteins that serve as analytes. Non-limiting example of lysing agentsinclude NP-40, CHAPS, deoxycholate, Triton X-100, NP40, and Tween 20.

In a further aspect, the reagent ball and/or the reservoir contains anRNAse inhibitor and/or DNAse inhibitor and/or protease inhibitor in anamount to inhibit RNAse, DNAse or protease activity native or endogenousto the sample being added for analysis.

Spacer elements can be advantageous because in some cases the primer canparticipate better in the amplification reaction if the nucleic acidportion of the primer is more distant from the label where othersterically hindering events could be occurring or have already occurredsuch as being bound to a particle or being bound to a signaling agent,both of which may be bulky. The spacer element separating the primerfrom a label comprises a polymer e.g., hexaethylene glycol, triethlyeneglycol, a C3 spacer phosphoramidite, a PC spacer, hexanediol and arecommercially available from Integrated DNA Technologies (seeidtdna.com/site/Catalog/Modifications/Category/6, last accessed Jul. 16,2016).

The sensor may be configured to be exposed to the mixed reservoir fluidand the sensor may be further configured to generate a signal indicativeof at least one of the presence, absence, or quantity of the sampletarget analyte within the sample. For example, particles from the mixedreservoir fluid comprising signaling agents may localize in an analysischannel over the sensor and the localized signaling agents may reactwith substrates from a substrate solution to generate electrical signalssensed by the sensor. The sensor may send the signal indicative of atleast one of the presence, absence, or quantity of the sample targetanalyte within the sample using the electrical signals from thereaction.

The reservoir may be further configured to permit mixing of a pluralityof solid particles within the reservoir fluid. Each solid particle maybe pre-bound to an affinity molecule of the plurality of affinitymolecules. The plurality of solid particles may be formed ofmagnetically responsive material and/or formed of non-magneticallyresponsive material. The non-magnetically responsive material may begold nanoparticles. The plurality of sample target analytes pre-bound tosample binding molecules may include 25-Hydroxy vitamin D3 and/or25-hydroxy vitamin D2 molecules pre-bound to binding protein molecules.The magnetically responsive material may be magnetically held over thesensor.

In accordance with yet another aspect, a sample analysis cartridge isprovided. The sample analysis cartridge may include an input tunnel, areservoir, a shuttle, and/or a collet, each of which may be within ahousing of the cartridge. The input tunnel may extend from an apertureand be configured to permit insertion of a sample collection devicehaving a distal portion adapted to be exposed to a sample. The reservoirmay be configured to hold a fluid. The shuttle may be disposed in theinput tunnel between the reservoir and the aperture in a first position.The collet may be disposed in the input tunnel and coupled to theshuttle in the first position. The collet may decouple from the shuttleduring insertion of the sample collection device in the input tunnel.The shuttle may move within the input tunnel from the first position toa second position after the collet is decoupled from the shuttle suchthat the shuttle is at least partially disposed within the reservoir inthe second position.

The sample analysis cartridge may include a sensor configured to beexposed to the fluid mixed with the sample. The sensor may generate asignal indicative of at least one of the presence, absence, or quantityof the one or more analytes within the sample. The shuttle may have afirst end and a second end disposed proximal to the first end in theinput tunnel. The second end of the shuttle may be configured to bedisposed within a lumen of the collet in the first position. The firstend of the shuttle may form a wall of the reservoir in the firstposition.

The collet may have one or more locking arms configured to couple thecollet to the shuttle in the first position. The one or more lockingarms may be configured to be deflected to decouple the one or morelocking arms from the shuttle responsive to a force applied on the oneor more locking arms by the sample collection device during insertion ofthe sample collection device in the input tunnel.

The sample analysis cartridge may include a sealing material configuredto fluidicly seal the fluid within the reservoir and a seal piercerdisposed partially within the input tunnel. The seal piercer may beconfigured to be contacted by the sample collection device within theinput tunnel and to move, responsive to force applied by the samplecollection device, to pierce the sealing material to vent the fluid inthe reservoir. The collet may have a slot and a portion of the sealpiercer may extend through the slot into the input tunnel to permitcontact between the seal piercer and the sample collection device.

The sample analysis cartridge may include a contact switch. The colletmay have a deflector portion disposed adjacent the contact switch andconfigured to deflect to activate the contact switch responsive to aforce applied on the deflector portion by the sample collection deviceduring insertion of the sample collection device in the input tunnel.The deflector portion of the collet may include an arm configured todeflect downward to activate the contact switch. The contact switch maybe positioned in the input tunnel such that activation of the contactswitch indicates full insertion of the sample collection device in theinput tunnel.

The shuttle may be configured to house a reagent ball comprisingreagents between first and second ends of the shuttle. Preferably, thereagent ball is not exposed to the fluid in the reservoir in the firstposition and is exposed to the fluid in the reservoir in the secondposition.

In accordance with yet another aspect, a sample analysis cartridge isprovided. The sample analysis cartridge may include an input tunnel, areservoir, a collet, and/or a contact switch, each of which may bewithin a housing of the cartridge. The input tunnel may extend from anaperture and may permit insertion of a sample collection device having adistal portion adapted to be exposed to a sample. The reservoir may beconfigured to hold a fluid. The collet may be disposed in the inputtunnel between the reservoir and the aperture. The collet may have adeflector portion and a lumen sized to receive the distal portion of thesample collection device therein. The contact switch may be disposedadjacent the deflector portion of the collet. The deflector portion maybe configured to deflect to activate the contact switch responsive to aforce applied on the deflector portion by the sample collection deviceduring insertion of the sample collection device in the input tunnel.

The sample analysis cartridge may include a shuttle disposed within theinput tunnel and configured to house a reagent ball(s) comprisingreagents between first and second ends of the shuttle. The deflectorportion of the collet may be an arm configured to deflect downward toactivate the contact switch. The contact switch may be positioned suchthat activation of the contact switch indicates full insertion of thesample collection device in the input tunnel.

The sample analysis cartridge may include a sensor configured to beexposed to the fluid mixed with the sample. The sensor may generate asignal indicative of at least one of the presence, absence, or quantityof the one or more analytes within the sample.

In accordance with another aspect, a sample analysis cartridge isprovided. The sample analysis cartridge may include an input tunnel, areservoir, a shuttle, and/or a sonicator, each of which may be within ahousing of the cartridge. The input tunnel may extend from an apertureand may permit insertion of a sample collection device having a distalportion adapted to be exposed to a sample. The reservoir may beconfigured to hold a fluid. The shuttle may define a first compartmentand a second compartment. The first and second compartments may beconfigured to be disposed within the reservoir in a mixing position. Thesonicator may be configured to emit acoustic waves to move the fluid inthe reservoir in a wave pattern between the first and secondcompartments to mix the fluid in the reservoir.

The shuttle may include a compartment divider configured to divide thefirst compartment from the second compartment. The compartment dividermay be a flange. Fluid flowing around the compartment divider mayfacilitate formation of the wave pattern. The compartment divider mayhave a slot configured to permit the fluid to flow through thecompartment divider via the slot during mixing. The first compartmentmay be a reagent ball compartment configured to house a reagent ballincluding reagents and the second compartment may be a samplecompartment configured to receive the sample from the sample collectiondevice, e.g., expelled from the sample collection device and/or on thedistal portion of the sample collection device. The reagent ball mayinclude, as described above, reagents for the amplification of a targetnucleic acid, e.g., polymerases, primers, and signaling agents. Thefirst and second compartments are preferably not disposed within thereservoir in a pre-mixing position.

The sonicator may be a piezoelectric transducer such as a piezoceramicdisc. The sonicator may form a wall of the reservoir, e.g., part of thebottom wall of the reservoir. The reservoir may be symmetric. Each ofthe walls of the reservoir may meet at an angle greater than apredetermined angle such as 60° to facilitate fluid emptying through anoutlet of the reservoir. The sonicator may be positioned off-center ofthe reservoir to facilitate mixing of the fluid within the reservoir.

The sample analysis cartridge may include a printed circuit boardcoupled to the sonicator via one or more spring contacts. The sonicatormay be electrically coupled to the printed circuit board only via theone or more spring contacts. The sonicator may be activated responsiveto a signal from a processor, e.g., the processor of the reader.

The sample analysis cartridge may include a temperature sensorconfigured to sense temperature indicative of temperature of the fluidin the reservoir. The temperature sensor may be disposed on a printedcircuit board positioned adjacent the sonicator.

The sample analysis cartridge may include a contact switch configured toindicate insertion of the sample collection device in the input tunnel.The sonicator may be configured to emit the acoustic waves afteractuation of the contact switch. For example, the reader may direct thesonicator to emit the acoustic waves after the reader receives anelectrical signal indicating that the contact switch has been activated.

The acoustic waves emitted by the sonicator may be configured toisothermally amplify reactions of the fluid mixed in the reservoir.

In accordance with another aspect, a method for isothermal amplificationof a target nucleic acid if present in a sample analysis cartridge isprovided. The method comprises or alternatively consists of contactingin the reservoir a reagent ball as described above and containing aplurality of reagents pre-selected for the amplification and detectionof the target nucleic acid with the sample to produce anamplicon-signaling agent complex coupled to the solid particle. Theamplicon comprises a nucleic acid duplex comprising: a reverse primercomplex comprising a nucleic acid comprising the target nucleic acidcoupled to a spacer element that in turn is coupled to a signaling agentand a forward primer complex comprising a nucleic acid comprising thetarget sequence coupled at one end to a capture element. In a furtheraspect, the reverse primer complex further comprises a signalingaffinity element conjugated to the signaling agent and the spacerelement. The amplicon-signaling agent complex in turn is conjugated toan affinity element on the solid particle that in turn, can be bound orheld to the sensor surface over a magnetic field. The amplicon-signalingagent complex in turn is conjugated to an affinity element on the solidparticle that in turn, can be held to the sensor surface over a magneticfield. If the analyte is present, the sensor detects and/or quantifiesthe signaling agent-labeled amplicon.

A sonicator may emit acoustic waves toward the reservoir to promoteamplification of the target nucleic acid in the reservoir. As notedabove, the amplicon-signaling agent complex may be reacted with asubstrate from a substrate reservoir. For example, the reaction mayoccur over a sensor in an analysis channel. A signal indicative of atleast one of a presence, absence or quantity of amplified nucleic acidmay be generated. The signal may be transmitted from the cartridge toanother device such as a reader.

A reagent ball may be held in a shuttle. The shuttle may be disposed inan input tunnel of the cartridge. The reagent ball may comprise reagentsfor amplification of a target nucleic acid by an isothermal reaction asnoted above. The reagents may comprise a polymerase, primers foramplification of the target nucleic acid and a signaling agent for thedetection of the amplification of the target nucleic acid. One or moreaffinity molecules may be covalently or non-covalently bound to a solidparticle for detection of the target nucleic acid.

In accordance with another aspect, a kit is provided. The kit mayinclude a reservoir, a sonicator, a temperature sensor, and/or aprocessor. The reservoir may be configured to hold a fluid and toreceive a sample collected by a sample collection device. The sonicatormay be configured to emit acoustic waves to mix the fluid and the samplein the reservoir. The temperature sensor may be configured to generate asignal indicative of temperature of the fluid in the reservoir. Theprocessor may be configured to activate the sonicator to emit theacoustic waves and to monitor the signal from the temperature sensor.The processor further may be configured to modify emission of theacoustic waves from the sonicator if the signal indicates a temperatureof the fluid in the reservoir outside a threshold. A sample analysiscartridge may include the reservoir, the sonicator, and/or thetemperature sensor, each of which may be within a housing of thecartridge, and a reader may include the processor. The reader may beconfigured to be electrically coupled to the sample analysis cartridge.

The sample analysis cartridge may include a printed circuit board andthe temperature sensor may be disposed on the printed circuit boardpositioned adjacent the sonicator. The sample analysis cartridge mayinclude a contact switch configured to generate a signal to indicateinsertion of the sample collection device in an input tunnel of thesample analysis cartridge. The processor of the reader may be configuredto receive the signal from the contact switch and to activate thesonicator after receipt of the signal from the contact switch. Theprocessor may modify emission of the acoustic waves from the sonicatorby lowering a duty cycle of the sonicator if the signal indicates thetemperature of the fluid in the reservoir is above the threshold. Theprocessor may modify emission of the acoustic waves from the sonicatorby increasing a duty cycle of the sonicator if the signal indicates thetemperature of the fluid in the reservoir is below the threshold. Theprocessor may modify emission of the acoustic waves from the sonicatorby deactivating the sonicator if the signal indicates the temperature ofthe fluid in the reservoir is above the threshold.

The acoustic waves emitted by the sonicator may be configured toisothermally amplify reactions of the fluid mixed in the reservoir. Thesample analysis cartridge may include a reagent ball disposed within thesample analysis cartridge. The sonicator may be configured to emit theacoustic waves to mix the fluid, the reagent ball, and the sample in thereservoir. The reagent ball may include polymerases, primers, andsignaling agents. The sample analysis cartridge may include a shuttleconfigured to house the reagent ball.

In accordance with another aspect, a sensor for use in a microfluidiccartridge is provided. The sensor may include a positive control workingelectrode, a working electrode, and/or a negative control workingelectrode. The positive control working electrode may include affinitymolecules pre-bound to the positive control working electrode. Forexample, the affinity molecules may be pre-bound to a surface of thepositive control working electrode disposed within an analysis channelof the cartridge. The positive control working electrode may beconfigured to generate a first signal based on a reaction betweensignaling agents directly or indirectly bound to the affinity moleculesand a chemical substrate. The signaling agents may be from a reagentball(s). The chemical substrate may be from fluid stored in thesubstrate reservoir. The working electrode may be configured to generatea second signal based on a reaction between the signaling agentslocalized at the working electrode and the chemical substrate. Thenegative control working electrode may include a self-assembledmonolayer. For example, the self-assembled monolayer may be at a surfaceof the negative control working electrode disposed within an analysischannel of the cartridge. The negative control working electrode may beconfigured to generate a third signal based on a reaction between thesignaling agents localized at the negative working electrode and thechemical substrate. As should be understood, “first”, “second”, and“third” differentiate terms and do not necessarily mean order.

The second signal may be indicative of at least one of the presence,absence, or quantity of one or more analytes within a sample. The firstsignal may be indicative of reliability of a test. For example, the testmay be determined to be reliable if the first signal indicates aquantity of the reaction within a predetermined range. The third signalis indicative of reliability of a test. For example, the test may bedetermined to be reliable if the third signal indicates a quantity ofthe reaction is below a threshold.

A cartridge may include the sensor. The cartridge may have an analysischannel and the positive control working electrode, the workingelectrode, and the negative control working electrode may be disposed inthe analysis channel.

A kit including the cartridge is also provided. The kit may include aprocessor configured to process the second signal to generateinformation indicative of at least one of the presence, absence, orquantity of one or more analytes within a sample. The processor mayprocess the first signal to determine if the first signal indicates aquantity of the reaction within a predetermined range. The processor maygenerate an alert if the quantity is outside the predetermined range.The processor may process the third signal to determine if the thirdsignal indicates a quantity of the reaction below a threshold. Theprocessor may generate an alert if the quantity is above the threshold.The processor may be a component of a reader.

The working electrode may be masked with a plurality of striationsconfigured to promote homogenous distribution of a plurality of magneticparticles directly or indirectly bound to the signaling agents localizedover the working electrode and to promote resistance to movement of theplurality of magnetic particles off of the working electrode.

The working electrode may include a self-assembled monolayer. Forexample, the self-assembled monolayer may be at a surface of the workingelectrode disposed within an analysis channel of the cartridge.

The working electrode may include affinity molecules pre-bound to theworking electrode. For example, the affinity molecules may be pre-boundto a surface of the working electrode disposed within an analysischannel of the cartridge.

Such methods and devices may be used, for example, to determine: fromwhich illness, among many, a person is suffering; to which drug orpoison, among many, a person is adversely reacting; or which chemical,among many, has contaminated the water. Other examples includequantifying the concentrations of various agents, that include withoutlimitation vitamins, hormones, proteins, or other analytes of interestwithin one's body, waterborne and foodborne pathogens, microbial growthand/or contamination of medical equipment, and other potentialdisease-causing contaminants from pets and livestock. Examples ofcontaminates include, but are not limited to viral, bacterial, andfungal pathogens, bloodstream infection (BSI), pneumonia (e.g.,ventilator-associated pneumonia [VAP]), urinary tract infection (UTI),and surgical site infection (SSI), Staphylococcus aureus, Methicillinresistant Staphylococcus aureus, Candida albicans, Pseudomonasaeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia,Clostridium difficile, Tuberculosis, Gastroenteritis,Vancomycin-resistant Enterococcus, Legionnaires' disease, Puerperalfever, MRSA, and E. coli. Examples of foodborne pathogens include, butare not limited to shigella, salmonella, vibrio, Yersinia, Listeria,Escherichia coli, and Campylobacter. The application of this technologyis not limited to pathogens or analytes that are important to the healthof human patients but also includes the health and maintenance of petsand livestock, e.g., veterinary uses.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are described below with reference to theaccompanying drawings, wherein like numerals denote like elements. Inthe drawings:

FIGS. 1A-1B provide schematic depictions of an exemplary analytedetection system for analyzing the presence, absence, and/or quantity ofone or more target analytes within a collected sample and for viewinganalysis results, wherein FIG. 1A shows components uncoupled and FIG. 1Bshows components coupled for analysis and charging.

FIG. 1C provides a schematic depiction of another exemplary analytedetection system for analyzing the presence, absence, and/or quantity ofone or more target analytes within a collected sample and for viewinganalysis results, wherein a charger is not provided.

FIGS. 2A and 2B illustrate perspective views of an exemplary samplecollection device for use in the detection system.

FIG. 3 illustrates a perspective view of an exemplary cartridge devicefor use in the detection system.

FIGS. 4A and 4B illustrate perspective views of the cartridge devicewith the sample collection device locked therein for analysis of thecollected sample, wherein FIG. 4A shows the top surface of the cartridgedevice and FIG. 4B shows the bottom surface of the cartridge device.

FIG. 5 illustrates an exploded view of the exemplary cartridge deviceshowing internal components that may be within the cartridge housing.

FIGS. 6A, 6B, and 6C illustrate an exemplary circuit board and anexemplary layer that may be used within the housing of the cartridgedevice, wherein FIG. 6A depicts the circuit board, FIG. 6B depicts thelayer, and FIG. 6C depicts the layer disposed on and coupled to thecircuit board.

FIGS. 7A, 7B, and 7C illustrate another exemplary circuit board andanother exemplary layer that may be used within the housing of thecartridge device, wherein FIG. 7A depicts the circuit board, FIG. 7Bdepicts the layer, and FIG. 7C depicts the layer disposed on and coupledto the circuit board and an absorbent pad.

FIGS. 7D through 7F illustrate alternative exemplary sensors that may beused within the housing of the cartridge device.

FIG. 8A illustrates an exemplary internal component coupled to anexemplary circuit board via a layer positioned therebetween, all ofwhich may be disposed within the housing of the cartridge device.

FIGS. 8B and 8C show a close-up views of certain components of thecircuit board and a valve for use in the housing of the cartridgedevice.

FIG. 8D shows a close-up views of alternative components of the circuitboard and a valve for use in the housing of the cartridge device.

FIGS. 9A and 9B illustrate exemplary shuttles that may be disposed inthe housing of the cartridge device, wherein the shuttles are each shownhousing a reagent ball.

FIG. 10A is a cross-sectional perspective view showing a samplecollection device partially inserted within an input tunnel of acartridge device.

FIG. 10B is a cross-sectional perspective view showing a tip of thesample collection device entering a shuttle disposed within the inputtunnel of the cartridge device.

FIG. 10C is a top view showing an exemplary orientation of piercingelements over reservoirs within the housing of the cartridge device.

FIG. 10D is a cross-sectional perspective view showing engagementbetween the sample collection device and a slider of a seal piercerwithin the input tunnel of the cartridge device, wherein part of thecartridge device is removed.

FIG. 10E is a cross-sectional side view showing engagement between thesample collection device and the seal piercer and sealing between thesample collection device and the shuttle, wherein the sample preparationreservoir remains sealed by the shuttle.

FIG. 10F is a top view showing engagement between the sample collectiondevice and the seal piercer in a pre-venting position.

FIG. 10G is a cross-sectional perspective view showing the piercingelement and the slider of the seal piercer in a pre-venting position,wherein the sealing material over the sample preparation reservoirwithin the cartridge device has not yet been pierced.

FIG. 10H is a cross-sectional side view illustrating transition from thepre-mixing and pre-venting positions towards the mixing and ventingpositions within the input tunnel of the cartridge device.

FIG. 10I is a top view showing movement of the sample collection devicecausing movement of the seal piercer to the venting position.

FIG. 10J is a cross-sectional perspective view showing the piercingelement piercing the sealing material over the sample preparationreservoir within the cartridge device.

FIG. 10K is a cross-sectional side view showing the sample collectiondevice in the venting and mixing positions, wherein the sealing materialover the sample preparation reservoir is vented and the collected sampleand the reagent ball are mixed within the fluid in the samplepreparation reservoir which has been re-sealed by the shuttle.

FIG. 10L is a cross-sectional perspective view showing the samplecollection device in the venting and mixing positions within thecartridge device.

FIG. 10M is a cross-sectional perspective view showing the samplecollection device in the venting and mixing positions within the samplepreparation reservoir of the internal component of the cartridge device.

FIGS. 10N, 10O, and 10P are cross-sectional side views showing enhancedmixing of the fluid in the sample preparation reservoir with thecollected sample and the reagent ball via a sonicator element.

FIGS. 11A through 11E illustrate an alternative seal piercer whereininsertion of a sample collection device within the input tunnel of thecartridge device also activates a switch to represent proper samplecollection device insertion.

FIGS. 12A through 12E illustrate cross-sectional side views of analternative sample collection device and an alternative cartridge devicefor collecting and analyzing a fluid sample.

FIG. 13A illustrates an exploded view of another exemplary cartridgedevice showing internal components that may be within the cartridgehousing.

FIGS. 13B through 13SS illustrate various views of exemplary samplecollection devices that may be used in the detection system.

FIGS. 14A through 14D illustrate exemplary collets that may be disposedin the housing of the cartridge device, wherein FIGS. 14A and 14C showperspective views and FIGS. 14B and 14D show cross-sectional views ofthe collets in FIGS. 14A and 14C, respectively.

FIG. 15A illustrates a cross-sectional view through the center of theinput tunnel of an exemplary cartridge in the pre-mixing, pre-venting,storage position.

FIG. 15B illustrates a cross-sectional view of the exemplary cartridgeand an exemplary sample collection device fully inserted in the inputtunnel in the mixing, venting, analysis position.

FIGS. 15C and 15D illustrate perspective views of the exemplarycartridge having the exemplary sample collection device inserted thereinin the pre-venting position (FIG. 15C) and in the venting position (FIG.15D).

FIGS. 16A through 16E illustrate cross-sectional side views showinginsertion of the sample collection device in the cartridge.

FIGS. 16F and 16G illustrate cross-sectional top views showing furtherdistal insertion of the sample collection device in the cartridge.

FIGS. 16H through 16J illustrate cross-sectional side views showingfurther distal insertion of the sample collection device in thecartridge, wherein the sample collection device is fully inserted in theinput tunnel in the mixing, venting, analysis position in FIG. 16J.

FIGS. 17A through 17D illustrate various views of an exemplary sonicatorelectrically coupled to a circuit board via spring contacts for usewithin an exemplary cartridge housing.

FIGS. 18A and 18B are cross-sectional side and top views, respectively,of another exemplary cartridge device.

FIG. 19 illustrates an exemplary process for monitoring temperatureduring enhanced mixing via the sonicator.

FIG. 20 is a graph showing measured temperature over time duringenhanced mixing via the sonicator.

FIG. 21A illustrates a perspective view of an exemplary reader devicefor use in the detection system.

FIG. 21B illustrates an exploded view of the exemplary reader device ofFIG. 21A showing internal components that may be within the readerhousing.

FIG. 22A shows a cross-sectional perspective view of the exemplaryreader device.

FIG. 22B is a cross-sectional side view showing a cartridge device(having a sample collection device partially inserted therein) partiallyinserted within the exemplary reader device.

FIGS. 22C and 22D are cross-sectional perspective and side views,respectively, showing the cartridge device inserted within the exemplaryreader device in the analysis position.

FIGS. 23A and 23B are graphs showing magnetic field strengths over thelength of a single working electrode for a single magnet (FIG. 23A)versus a dual magnet (FIG. 23B) design.

FIG. 24 provides a flowchart of one embodiment of a method for detectingthe presence, absence, and/or quantity of one or more target analytes ina sample.

FIG. 25A illustrates a perspective view of an exemplary charger that maybe used in the detection system.

FIG. 25B illustrates an exploded view of the exemplary charger of FIG.25A showing internal components that may be within the charger housing.

FIGS. 26A and 26B provide schematic depictions of molecules andreactions found within one embodiment of the presently disclosed analytedetection system.

FIGS. 26C and 26D provide schematic depictions of molecules andreactions found within another embodiment of the presently disclosedanalyte detection system.

FIGS. 27A and 27B provide schematic depictions of molecules andreactions found within yet another embodiment of the presently disclosedanalyte detection system.

FIG. 28A is a schematic depiction of molecules within a sample on asample collection device.

FIG. 28B is a schematic depiction of molecules within two reagent ballsfor reacting with the molecules within the collected sample.

FIG. 28C is a schematic depiction of molecules within a single reagentball for reacting with the molecules within the collected sample.

FIG. 28D is a schematic depiction of molecules showing the collectedsample being introduced to a sample preparation reservoir.

FIG. 28E is a schematic depiction of molecules showing mixing of themolecules of the collected sample with sample preparation reagentmolecules within the fluid of the sample preparation reservoir.

FIGS. 28F, 28G, and 28H are schematic depictions of molecules showingreactions between the molecules of the collected sample and the samplepreparation reagent molecules within the fluid of the sample preparationreservoir.

FIG. 29A is a graph showing electrochemical sensor readings versusconcentration of a target analyte using pre-bound competitor bindingmolecules and FIG. 29B shows a graph comparing electrochemical sensorreadings versus concentration when a competitor binding molecule is notpre-bound.

FIG. 30A is another schematic depiction of molecules within a sample ona sample collection device.

FIG. 30B is another schematic depiction of molecules within two reagentballs for reacting with the molecules within the collected sample.

FIG. 30C is another schematic depiction of molecules within a singlereagent ball for reacting with the molecules within the collectedsample.

FIG. 30D is another schematic depiction of molecules showing thecollected sample being introduced to a sample preparation reservoir.

FIG. 30E is another schematic depiction of molecules showing mixing ofthe molecules of the collected sample with sample preparation reagentmolecules within the fluid of the sample preparation reservoir.

FIGS. 30F, 30G, and 30H are schematic depictions of molecules showingreactions between the molecules of the collected sample and the samplepreparation reagent molecules within the fluid of the sample preparationreservoir.

FIGS. 31A through 32H show an exemplary process for detecting thepresence, absence, and/or quantity of a target analyte(s) within asample in a cartridge.

FIGS. 32A through 32K show an exemplary process for detecting thepresence, absence, and/or quantity of a target analyte(s) within asample in a cartridge using isothermal amplification.

FIG. 33 provides a schematic depiction of the exemplary analytedetection system FIGS. 1A-1B communicatively coupled to one or moreservers via a network.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

In the following detailed description, reference is made to theaccompanying drawings, which form part of the present disclosure. Theembodiments described in the drawings and description are intended to beexemplary and not limiting. As used herein, the term “exemplary” means“serving as an example or illustration” and should not necessarily beconstrued as preferred or advantageous over other embodiments. Otherembodiments may be utilized and modifications may be made withoutdeparting from the spirit or the scope of the subject matter presentedherein. Aspects of the disclosure, as described and illustrated herein,can be arranged, combined, and designed in a variety of differentconfigurations, all of which are explicitly contemplated and form partof this disclosure.

Various devices, systems, kits, and methods disclosed herein areintended to isolate, tag, and detect a target analyte within a sampletaken from a specimen. In certain embodiments, chemical reactions areemployed to enable such detection.

Various embodiments of systems described herein are designed to create aself-contained environment in which any of the chemical reactions occurin an automated manner entirely or substantially without humanintervention, for example, as described in commonly assigned U.S. PatentPub. No. 2014/0336083 to Khattak, U.S. Pat. No. 9,034,168 to Khattak,U.S. Pat. No. 9,052,275 to Khattak, U.S. Pat. No. 9,086,417 to Khattak,U.S. Pat. No. 9,207,244 to Khattak, U.S. Pat. No. 9,207,245 to Khattak,and U.S. Pat. No. 9,360,491 to Sever, the entire contents of each ofwhich are incorporated herein by reference. In some designs describedherein, one or more chemical reactions proceed without any need for anoperator to add or remove reagents from the system. In certainembodiments, the systems are closed such that biohazard risks, such asthe risk of spilling sample collected from a specimen, are minimized. Invarious embodiments, such systems include at least, a sample collectiondevice, a cartridge device, and a reader device. Some exemplaryembodiments of such devices are described in detail below.

FIGS. 1A and 1B illustrate an exemplary analyte detection systemconstructed in accordance with the principles of the present disclosure.Detection system 100 may include sample collection device 200, cartridgedevice 300, reader device 400, charger 500, and/or software-baseddetection interface system 600. Detection system 100 may be used todetect the presence, absence, and/or quantity of one or more targetanalytes.

Sample collection device 200 is configured to be exposed to a sample foranalysis. For example, sample collection device 200 may be exposed to abiological sample, such as, but not limited to, blood, plasma, urine,saliva, mucous, cellular material and/or other biological material fordetermining the presence, absence, and/or quantity of one or more targetanalytes within the sample. In addition or alternatively, the samplecollection device is exposed to a solid or other surface that issuspected of harboring a target analyte, e.g., a food-borne pathogen andthe surface is a cooking or food preparation surface.

Cartridge device 300 is configured to analyze the sample collected withsample collection device 200. Cartridge device 300 may include inputtunnel 301 that extends from aperture 302 into the cartridge housing.Input tunnel 301 is configured to permit insertion of sample collectiondevice 200 as shown in FIG. 1B such that the collected sample may beanalyzed within cartridge device 300. Based on the analysis, cartridgedevice 300 is configured to generate electric signals indicative of thepresence, absence, and/or quantity of one or more target analytes withinthe sample.

Reader 400 is configured for electric coupling with cartridge device 300to permit transmission of the electric signals indicative of thepresence, absence, and/or quantity of one or more target analytes withinthe sample generated by cartridge device 300. Cartridge device 300 maybe electrically coupled to reader 400 by inserting cartridge device 300within reader opening 401 of reader 400 as shown in FIG. 1B such thatrespective electrical connectors of cartridge device 300 and reader 400contact one another. Reader 400 may comprise a computer readable mediumwith instructions that, when executed by a processor of reader 400,cause electrical components of cartridge 300 to perform steps foranalyzing the sample on sample collection device 200. Preferably, theinstructions are not executed until cartridge device 300 is electricallycoupled to reader 400 and sample collection device 200 is suitablydisposed within cartridge device 300, for example, as shown in FIG. 1Bor 4A.

In one embodiment, sample collection device 200 and cartridge device 300are each disposable and designed for one time use while reader 400 isdesigned for multi-use and for receiving many different cartridgedevices throughout the life of reader 400 such that many samples areanalyzed by reader 400 for determining the presence, absence, and/orquantity of one or more target analytes within the respective samples.Such a configuration is expected to promote sanitary use of the system,as the components exposed to the sample are disposable, while reducingcosts as the components with more expensive electronics, e.g., reader400, may be used repeatedly.

Charger 500 is configured to charge one or more batteries within reader400, e.g., via respective inductive coils disposed within the housingsof charger 500 and reader 400. Charger 500 may be plugged into aconventional socket, e.g., via a cord or a cord with an AC to DC powerconverter, for charging components within charger 500 to permit chargingof reader 400.

As will be readily apparent to one skilled in the art, the detectionsystem need not require a charger. For example, referring to FIG. 1C,detection system 100′ is constructed similarly to detection system 100of FIGS. 1A and 1B, wherein like components are identified bylike-primed reference numbers. Thus, for example, cartridge device 300′in FIG. 1C corresponds to cartridge device 300 of FIGS. 1A and 1B, etc.As will be observed by comparing FIGS. 1C and 1B, detection system 100′does not include charger 500. In such an embodiment, reader 400′ may beplugged into a conventional socket, e.g., via a cord or a cord with anAC to DC power converter, for powering components of reader 400′ and/orreader 400′ may include a suitable battery such as a replaceable batteryor rechargeable battery and reader 400′ may include circuitry forcharging the rechargeable battery, and a detachable power cord.

In FIGS. 1A and 1B, software-based detection interface system 600 isinstalled and runs on computing device 601 to permit a user to reviewanalyte detection test results, e.g., on display 602 of computing device601. Computing device 601 may be, for example, a smartphone, smartwatch,tablet, wearable device, a laptop or other computer. As shown in FIGS.1A and 1B, reader 400 may communicate with computing device 601wirelessly to transmit data indicative of the presence, absence, and/orquantity of one or more target analytes based on the electrical signalsgenerated within cartridge device 300. In addition or alternatively, aremovable wired connection, such as a cable connection, may be providedbetween reader 400 and computing device 601. Software-based detectioninterface system 600 may comprise a computer readable medium withinstructions that, when executed by a processor of computing device 601,cause display 602 to display information indicative of the presence,absence, and/or quantity of one or more target analytes.

Sample Collection Devices and Cartridges

The sample collection device of various embodiments is configured tocollect a sample from a specimen. Sample collection devices may beconfigured to collect cells and other biological material from anydesired region or location, for example, an inner cheek, the throat, anasal passageway, an ear, from urine, from blood, from plasma, fromsaliva, or from another body part. One exemplary sample collectiondevice includes a unit that wicks a small droplet of blood or urine intoa small capillary channel. In other embodiments, the sample collectiondevice may be configured to collect biological material, particulates,or other chemicals from the environment, such as, for example, from theair or the water, or from a physical surface or other structure.

The sample collection device of various embodiments is sized and shapedto collect a sufficiently large sample from an appropriate location of aspecimen such that it is possible, using the other devices describedbelow, to detect the presence, absence, and/or quantity of one or moretarget analytes in and/or on the specimen. For example, for some targetanalytes, such as ones associated with a virus causing cold or flu-likesymptoms, the sample collection device may be a nose-insertion swab; theswab is sized and shaped to collect a sufficient amount of sample from anasal passageway of an individual to enable detection of target analytesassociated with the virus causing cold or flu-like symptoms, if presentin the individual. For other target analytes, such as, for example, onesassociated with strep throat, the sample collection device may be athroat swab shaped to scrape sufficient cells from an individual'sthroat or mouth. As another example, the sample collection deviceappropriate for collecting a target analyte associated with HIV maycomprise a blood lancet. In another example, a sample collection deviceconfigured to collect urine may be appropriate for collecting targetanalytes for various tests, including, for example, tests for trackingtestosterone levels, drug levels, vitamin levels, and/or fertility. Asample collection device for collecting fluid, such as urine, blood,plasma, or saliva, may include features for compressing a wickingportion of the device to expel sample absorbed on the wicking portionfor analyzing the expelled sample. In yet a further aspect, the samplecollection devise is shaped to collect a sample from a solid surface,e.g., on a medical device, on medical equipment or from the surface of afood preparation surface such as a cutting board or flat surface.

Referring to FIGS. 2A and 2B, sample collection device 200 isillustrated. Sample collection device 200 is configured to collect asmall quantity of a sample to be analyzed and configured for full orpartial insertion within cartridge device 300 after sample collection.Sample collection device 200 may include distal portion 201, proximalportion 202, and shaft 203 extending therebetween. Distal portion 201may include tip 204 having tube 205 therein. Sample collection device200 also may include handle 206, proximal sealing zone 207, distalsealing zone 208, and/or engagement zone 209.

Distal portion 201, including tip 204, is configured to be exposed to asample such that, at most, a predetermined volume of the sample isdisposed in tube 205 for analysis. Collection of a predetermined volumeof the sample is expected to promote accuracy of analyte analysis as asubstantially known quantity of the sample will be analyzed. Tip 204 maybe transparent to permit a collector to verify that sample is disposedin tube 205. Tip 204 may have a rounded end as illustrated althoughvarious shapes may be used including any blunt or substantially blunttip shape. Tip 204 may be configured to collect a sample from anydesired region or location, for example, an inner cheek, the throat, themouth, a nasal passageway, an ear, from urine, from blood, from plasma,from saliva, or from another body part.

Proximal portion 202 may include handle 206 sized and shaped to be heldby a collector's hand. Handle 206 may include gripping protrusions asillustrated. Handle 206 may further lock sample collection device 200within the input tunnel of cartridge device 300. Sample collectiondevice 200 also may include proximal sealing zone 207 configured forsealing the input tunnel of cartridge device 300 when sample collectiondevice 200 is inserted in the input tunnel. Proximal sealing zone 207may include a protrusion extending around shaft 203 and sized greaterthan the input tunnel opening so as to seal off the input tunnel. Assuch, the protrusion may further lock sample collection device withinthe input tunnel of cartridge device 300. Handle 206 may be breakable orotherwise removable from the remainder of sample collection device 200following insertion of the remainder of sample collection device 200into cartridge device 300.

Shaft 203 is elongated to facilitate easy and sanitary collection, witha collector's hand removed from the site of collection. For example,shaft 203 may be elongated such that tip 204 may be exposed to a samplewithin an inner cheek, the throat, the mouth, a nasal passageway, anear, from urine, from blood, from plasma, from saliva, etc. to collectfluid, cells, and other biological material, while handle 206 is notexposed to the sample. Shaft 203, tip 204, and handle 206 may be formedof the same material or of different materials. Shaft 203, tip 204, andhandle 206 may be formed of a plastic. Sample collection device 200 maybe pre-packaged within sterile packaging and is preferably configuredfor one-time use.

Sample collection device 200 may have distal sealing zone 208 forfacilitating the formation of a liquid-tight seal between samplecollection device 200 and cartridge device 300 after insertion of samplecollection device 200 into cartridge device 300. For example, distalsealing zone 208 may be sized and shaped to seal the collected sample ontip 204 and fluid within a sample preparation reservoir of cartridgedevice 300 within cartridge device 300. Distal sealing zone 208 may beof greater radial size than tip 204. For example, distal sealing zone208 may include a shoulder extending further from the longitudinal axisof sample collection device 200 than tip 204 such that shoulder abutsagainst a portion of cartridge device 300, e.g., a shuttle, to form theliquid-tight seal. In this manner, the sample may be sealed withincartridge device 300 to reduce leakage and exposure of the sampleoutside the cartridge. In addition, the shoulder may be used to move aseal piercer to vent one or more reservoirs within cartridge device 300before, during, or after formation of the liquid-tight seal.

Sample collection device 200 may include engagement zone 209 configuredfor engagement with one or more components of cartridge device 300. Forexample, engagement zone 209 may be configured to be coupled,permanently or temporarily, to a seal piercer of the cartridge device tomove the seal piercer within the cartridge device responsive to movementof sample collection device 200. Engagement zone 209 also may facilitatefixed engagement between sample collection device 200 and the cartridgedevice such that sample collection device 200 is mated irreversibly andimmovably with the cartridge when sample collection device 200 isinserted a predetermined distance in the input tunnel of the cartridge.Engagement zone 209 may be a groove around shaft 203 as illustrated ormay be multiple grooves extending a shorter distance from thelongitudinal axis than the regular shaft surface and/or may be one ormore protrusions extending a greater distance from the longitudinal axisof sample collection device 200.

In various embodiments, a cartridge is formed of a housing, whichdefines an enclosed space and has various features that enable thecartridge to do one or more of the following: receive a sample withtarget analytes from a sample collection device, store the sample withsample preparation reagents, provide a space for mixing and binding ofthe target analytes with sample preparation reagents, provide ananalysis zone wherein bound target analytes localize over sensors fordetection, provide a fluid medium for transporting the bound targetanalytes to the analysis zone, store and provide a substrate that canundergo a detectable reaction when introduced to the bound targetanalytes, provide a fluid medium for transporting the substrate to thebound target analytes in the analysis zone, and provide a wastecollection zone where waste is stored.

In various embodiments, the cartridge is a substantially closed systemwherein the reactions needed to detect the presence, absence, and/orquantity of one or more target analytes occur within the cartridge. Thecartridge of such embodiments is said to be “substantially closed”because the only inputs needed into the cartridge system are one or moreof the following: a sample from a specimen, energy to facilitate mixingand bound, and a magnetic force to facilitate localization of boundtarget analytes within an analysis zone; the only outputs from thecartridge are electrical signals. In various embodiments, the cartridgeis target-analyte-specific with the included sample preparation reagentsselected to detect one or more specific target analytes. Differentcartridge types include different reagents intended to identifydifferent target analytes. For example, different cartridge types mayinclude inflammation, influenza, testosterone, fertility, HIV, andVitamin D which each include application-specific reagents intended toidentify different target analytes.

Referring now to FIG. 3, an exemplary cartridge is illustrated.Cartridge device 300 may include input tunnel 301 that extends fromaperture 302 on front surface 303 into cartridge housing 304. Cartridgehousing 304 may have a substantially rectangular prism shape asillustrated, although the present disclosure is not limited thereto.Cartridge housing 304 has front surface 303, top surface 305, right sidesurface 306, left side surface 307 (shown in FIG. 4A), bottom surface308 (shown in FIG. 4B), and back surface 309 (shown in FIG. 4B).Cartridge housing 304 may be formed of a single component or multiplecomponents. For example, cartridge housing 304 may include first covercomponent 310 configured to be laterally coupled to second covercomponent 311 such that the internal components of cartridge device 300are housed therein.

FIGS. 4A and 4B illustrate sample collection device 200 inserted withininput tunnel 301 of cartridge device 300 in a mixing position. In themixing position, proximal sealing zone 207 of sample collection device200 may seal input tunnel 301 at aperture 302 to reduce or eliminateleakage from input tunnel 301. Input tunnel 301 may be integrally formedwith housing 304 of cartridge device 300 or may be detachably coupled tocartridge housing 304. As shown in FIG. 4B, cartridge device 300 mayinclude electrical connector 312 configured for electrical coupling withthe reader, e.g., via an electrical connector of the reader.Accordingly, signals indicative of the presence, absence, and/orquantity of one or more target analytes may be transmitted fromcartridge device 300 via electrical connector 312 to the reader.Electrical connector 312 may be positioned on bottom surface 308 andback surface 309 to facilitate coupling with the electrical connectorwithin the opening of the reader.

Bottom surface 308 of cartridge device 300 may include first rampportion 313, second ramp portion 314, and magnetic generator depression315. First ramp portion 313 is configured to gradually depress one ormore magnetic generators of the reader during insertion of cartridgedevice 300 within the opening of the reader. First ramp portion 313 maybegin in the depression of cartridge housing 304 where electricalconnector 312 is positioned and ramp down to bottom surface 308. Ascartridge device 300 is inserted past first ramp portion 313, themagnetic generators remain in a depressed position until contacting thesecond ramp portion 314 which ramps up into magnetic generatordepression 315. Second ramp portion 314 is configured to gradually guidethe one or more magnetic generators of the reader into magneticgenerator depression 315. Magnetic generator depression 315 is disposedbeneath one or more working electrodes of cartridge device 300 such thatthe one or more magnetic generators of the reader move up into magneticgenerator depression 315 and are disposed adjacent the one or moreworking electrodes when cartridge device 300 is fully inserted in thereader. Second ramp portion 314 also facilitates removal of cartridgedevice 300 from the reader by gradually depressing the one or moremagnetic generators of the reader during cartridge removal.

Referring now to FIG. 5, an exploded view of cartridge device 300 isshown. Cartridge device 300 may include internal component 316—which mayinclude sample preparation reservoir 317, wash reservoir 318, andsubstrate reservoir 319—sealing material 320, seal piercer 321—which mayinclude slider 322 and piercer 323—shuttle 324, desiccant 325, inputtunnel component 326, sonicator element 327, absorbent pad 328, layer329, analysis channel 330, and circuit board 331 electrically coupled tomemory 332. The internal components may be disposed within housing 304,e.g., between first and second cover components 310 and 311.Alternatively, one or more internal components may be disposed withinone housing while other internal components may be disposed withinanother housing(s). In the case of multiple housings, such separatehousing may be configured to couple to one another.

Internal component 316 is configured to define one or more reservoirs,illustratively sample preparation reservoir 317, wash reservoir 318, andsubstrate reservoir 319. Internal component 316 may further define aportion of analysis channel 330 such as by creating the upper boundaryof analysis channel 330 when cartridge device 300 is assembled. Internalcomponent 316 may house other internal components such as absorbent pad328. Further, internal component 316 may be formed of a suitablematerial, such as plastic, and may have a base sized in a generallyrectangular shape to sit over circuit board 331.

Sample preparation reservoir 317 is configured to hold a fluid,preferably a liquid having sample preparation reagents. For example, thefluid may be water, saline solution, water/saline solution mixed withone or more of magnetic particles, affinity molecules, connectionmolecules, signaling agents, competitor binding molecules, competitormolecules, labels, and/or signaling agents, as described in furtherdetail below. Sample preparation reservoir 317 is positioned adjacent tothe distal end of input tunnel 301 such that input tunnel 301 leads tosample preparation reservoir 317. As described further below, samplepreparation reservoir 317 may be partially formed with sonicator element327, e.g., as part or all of the bottom surface, which facilitatesmixing of the fluid and additional particles in the fluid. In addition,sample preparation reservoir 317 may be partially formed with an end ofshuttle 324 during the pre-mixing state and partially formed withanother portion of shuttle 324 during the mixing state when one or morereagent balls and the sample are disposed in sample preparationreservoir 317. In this manner, sample preparation reservoir 317 remainsfluidicly sealed in the pre-mixing state by shuttle 324 and in themixing state by shuttle 324 and continuously fluidicly sealed throughoutmovement from the pre-mixing state to the mixing state such that fluiddoes not leak proximally past shuttle 324. Sample preparation reservoir317 is positioned such that upon insertion of sample collection device200 into input tunnel 301, distal portion 201 having the sample, e.g.,at tip 204 and/or tube 205, enters sample preparation reservoir 317.When sample collection device 200 enters sample preparation reservoir317, sample preparation reservoir 317 becomes further filled with sampleparticles, including one or more target analytes, if present in thesample. The fluid may be gently mixed, e.g., via sonicator element 327,with the one or more reagent balls and the sample to suspend andhybridize particles within sample preparation reservoir 317. The targetanalytes in the sample may hybridize and/or bind, at least, to themagnetic particles and/or to the affinity molecules present among thesample preparation reagents forming magnetic particle-bound complexesand/or affinity molecule-target complexes. Sample preparation reservoir317 is configured to permit release, e.g., via an outlet, of the fluidhaving the sample and sample preparation reagents mixed therein intoanalysis channel 330 for analyzing the presence, absence, and/orquantity of one or more target analytes within the sample. The outlet ofsample preparation reservoir 317 may be sealed with a heat actuatedvalve. When the valve opens, fluid from sample preparation reservoir 317acts as a transport medium causing the magnetic particle-bound complexesand/or affinity molecule-target complexes and other particles to flowfrom sample preparation reservoir 317 into the analysis channel 330.Advantageously, the fluid serving as the mixing medium and storagemedium within sample preparation reservoir 317 also acts as the flowmedium to transport the contents of sample preparation reservoir 317 toan analysis zone within analysis channel 330 without the need for apump.

Wash reservoir 318 is configured to hold a fluid, preferably a liquidconfigured as a wash solution. Wash reservoir 318 is further configuredto permit release, e.g., via an outlet, of the wash solution intoanalysis channel 330 to move particles in the mixed fluid previouslyreleased from sample preparation reservoir 317 that are not bound to amagnetic particle or a pre-bound surface affinity molecule off a workingelectrode and/or off a positive control working electrode in theanalysis channel. The outlet of wash reservoir 318 may be sealed with aheat actuated valve. When the valve opens, the wash solution flows fromwash reservoir 318 into analysis channel 330, thereby removing all orsubstantially all unbound detector agents and/or unbound competitivebinding agents from analysis channel 330. In one aspect, most or allfree-floating, unbound molecules from sample preparation reservoir 317are washed from analysis channel 330 to reduce the likelihood of havingany non-specific binding of significance and/or non-specific signalgenerated by free floating signaling agents, e.g., HRP, of significanceoccur within an analysis zone of analysis channel 330.

Substrate reservoir 319 is configured to hold a fluid, preferably asubstrate solution comprising a substrate such as a chemical substrate.The fluid of substrate reservoir 319 may include a substrate thatundergoes a reaction in the presence of a signaling agent from samplepreparation reservoir 317. For example, the substrate of substratereservoir 319 may undergo an oxidation reaction in the presence of anoxidizing enzyme from sample preparation reservoir 317. The fluid may bea substrate solution including acceptor molecules, such as hydrogenperoxide, and the substrate which may be an enzyme substrate such asTetramethylbenzidine (TMB) and/or o-phenylenediamine dihydrochloride(OPD) molecules. As an example, the substrate may be a commerciallyavailable enzyme-linked immunosorbent assay (ELISA) substrate.Preferably, the substrate is oxidizable and/or reducible. The acceptormolecules may be configured to receive electrons stripped from thesubstrate by the signaling agent (thereby oxidizing the substrate)during the reaction between the substrate and the signaling agent. Forexample, when the acceptor molecules are hydrogen peroxide, an oxidasereaction between the substrate, e.g., TMB, OPD, and the signaling agent,e.g., HRP, SBP, causes electrons to be stripped from the substrate anddonated to the acceptor molecules (e.g., hydrogen peroxide) during theoxidase reaction such that the acceptor molecules convert to anothermolecule (e.g., water). In some embodiments, ferricyanide is used as thesubstrate (and reacted with a signaling agent, that may be an oxidationdye such as Methylene Blue, from sample preparation reservoir 317).Substrate reservoir 319 is further configured to permit release, e.g.,via an outlet, of the fluid with the substrate into analysis channel330. The outlet of substrate reservoir 319 may be sealed with a heatactuated valve. When the valve opens, fluid from substrate reservoir 319acts as a transport medium causing the chemical substrate to flow fromsubstrate reservoir 319 into analysis channel 330.

One skilled in the art will appreciate that while three reservoirs aredepicted, in various embodiments, the plurality of reservoirs mayinclude two reservoirs or four or more reservoirs and may adoptalternative spatial configurations. For example, wash reservoir 318 andsubstrate reservoir 319 could be combined into a reservoir configured tohold a fluid that acts as a wash solution and having chemicalsubstrates. In addition, while the reservoirs are preferably pre-filledwith the respective fluids described above, the disclosure is notlimited thereto and one or more reservoirs may be empty in the non-usestate and filled with the respective fluid during the mixing state.

Sealing material 320 is configured to fluidly seal the fluid in one ormore reservoirs. For example, sealing material 320 may fluidly seal therespective fluids in sample preparation reservoir 317, wash reservoir318, and substrate reservoir 319. Sealing material 320 may be a singlepiece of material configured to cover all reservoirs, as illustrated, ormay be separate pieces each configured to cover one or more reservoirswithin cartridge device 300. Sealing material 320 may be any materialthat can fluidly seal fluid, such as a foil. Preferably, sealingmaterial 320 is a liquid-impermeable membrane.

Seal piercer 321 is configured to pierce sealing material 320 to ventthe fluid in sample preparation reservoir 317, wash reservoir 318,and/or substrate reservoir 319. Seal piercer 321 may be configured to becontacted by distal portion 201, e.g., at a shoulder or engagement zone209, of sample collection device 200 within input tunnel 301 and to movewithin housing 304, responsive to force applied by sample collectiondevice 200, to cause sealing material 320 to be pierced to vent thefluid in sample preparation reservoir 317, wash reservoir 318, and/orsubstrate reservoir 319. Seal piercer 321 is disposed within housing 304and may be partially disposed within input tunnel 301. In oneembodiment, seal piercer 321 is configured to move in a first direction,e.g., laterally, responsive to insertion of sample collection device 200in input tunnel 301 and in a second direction, e.g., vertically, topierce into sealing material 320.

Seal piercer 321 may be a single piece or may include multiple pieces.Illustratively, seal piercer 321 includes slider 322 and piercer 323.Slider 322 is disposed within housing 304 and may be partially disposedwithin input tunnel 301. For example, slider 322 may have an engageradapted to be disposed within input tunnel 301. The engager may beconfigured to be temporarily or permanently coupled to sample collectiondevice 200, e.g., at a shoulder or at engagement zone 209, to permitmovement of slider 322 responsive to insertion of sample collectiondevice 200 into input tunnel 301. The engager may be sized to fit withina groove of engagement zone 209, e.g., U-shaped as illustrated, or toreceive a protrusion of engagement zone 209. Slider 322 may beconfigured to move within housing 304, responsive to force applied bysample collection device 200 resulting from a collector pushing samplecollection device distally into input tunnel 301, to cause sealingmaterial 320 to be pierced by piercer 323 to vent the fluid in samplepreparation reservoir 317, wash reservoir 318, and/or substratereservoir 319. Piercer 323 may be one or more piercing elements withends sufficiently sharp to cut open sealing material 320. As describedin detail below, piercer 323 may include three different piercers, eachdisposed within housing 304 above one of sample preparation reservoir317, wash reservoir 318, or substrate reservoir 319. As slider 322 moveswithin input tunnel 301 as caused by insertion of sample collectiondevice 200, slider 322 contacts piercer 323 and moves piercer 323 in adirection to pierce sealing material 320. In one embodiment, slider 322is configured to move in a first direction, e.g.,laterally/substantially parallel to movement of sample collection device200, responsive to insertion of sample collection device 200 in inputtunnel 301 to cause piercer 323 to move in a second direction, e.g.,vertically, to pierce into sealing material 320.

Shuttle 324 is configured to be disposed within housing 304, preferablybetween sample preparation reservoir 317 and aperture 302. For example,shuttle 324 may be disposed within input tunnel 301 when the cartridgeis in a non-use state such that a distal end of shuttle 324 forms a wallof sample preparation reservoir 317 to seal fluid therein. Shuttle 324may define one or more compartments configured to receive the collectedsample from sample collection device 200 when inserted in input tunnel301. Shuttle 324 also may define one or more additional compartmentsconfigured to house one or more reagents balls. Shuttle 324 may beconfigured to move within housing 304 when subjected to a thresholdforce, e.g., caused by contacting sample collection device 200 toshuttle 324, to a second position such that the one or more samplecompartments having the sample and/or the one or more reagent ballcompartments having the one or more reagent balls therein are disposedin the fluid within sample preparation reservoir 317. The proximal endof shuttle 324 may, in conjunction within sample collection device 200,re-form the wall of sample preparation reservoir 317 to seal fluidtherein when the sample and/or the one or more reagent ball are insample preparation reservoir 317. In this manner, sample preparationreservoir 317 remains fluidicly sealed in the non-use state by shuttle324 and in the mixing state by shuttle 324. In addition, unlike abreakable membrane housing reagents, shuttle 324 may remain intact asthe sample is moved into sample preparation reservoir 317 for analysis.

Desiccant 325 may be disposed within housing 304 and in fluidiccommunication with one or more reagent balls housed in shuttle 324.Desiccant 325 is configured to absorb moisture that enters into housing304 to reduce moisture exposure to the one or more reagent balls in thenon-use state. Desiccant 325 may be a pad and may be at least partiallydisposed within input tunnel 301. Desiccant 325 may have a lumen sizedto permit the sample collection device to be inserted therethrough.

Input tunnel component 326 forms a portion of input tunnel 301 and issized and shaped to secure shuttle 324 within input tunnel 301. Forexample, input tunnel component 326 may have a U-shape to house agenerally cylindrical shuttle.

Sonicator element 327 is disposed within housing 304 and preferablyadjacent to, or integral with, sample preparation reservoir 317 topermit mixing of the fluid therein. Sonicator element 327 is configuredto transmit controlled amounts of energy into sample preparationreservoir and may include piezoelectric components. Sonicator element327 may be disposed on or form a bottom wall of sample preparationreservoir 317. Sonicator element 327 may be electrically isolated, e.g.,via use of a relay, from other electrical components within housing 304such as the components on circuit board 331, including the sensor andthe heaters. Sonication energy may be controlled to achieve mixing andbinding of components within sample preparation reservoir 317 whilelimiting damage caused to fragile DNA probes or other molecules such asantibodies and enzymes. Sonicator element 327 may include apressure-sensitive piezoelectric disk. Sonicator element 327 also mayinclude a high water content blister disposed between the samplepreparation reservoir 317 and the piezoelectric disk. Such a high watercontent blister may be affixed under sample preparation reservoir 317 inthe cartridge production process. The high water content blister mayfacilitate delivery of sonic energy from sonicator element 327 to samplepreparation reservoir 317 with minimal attenuation. The blister may bereplaced with another appropriately conducting sonication medium and thecomponent serving as a sonication medium may be dry on the outside, withno liquid residue present.

Absorbent pad 328 is disposed within housing 304 at the downstream-mostend of analysis channel 330. Absorbent pad 328 wicks fluid from analysischannel 330, thereby encouraging fluid to flow downstream to absorbentpad 328. Absorbent pad 328 may act as a waste receptacle, collecting allwaste fluids and waste particles after they have flowed through analysischannel 330. The size and degree of absorbency of absorbent pad 328 maybe selected to meter the flow of fluids and particles within theanalysis channel 330. For example, the volume of fluid that absorbentpad 328 can wick must be great enough to drain all fluid from samplepreparation reservoir 317 and wash reservoir 318 and draw the fluidcarrying the chemical substrate from the substrate reservoir 319. Such acondition may serve as the lower limit of absorbency.

Layer 329 is disposed between internal component 316 and circuit board331 and forms part of analysis channel 330. Layer 329 may be an adhesivelayer configured to couple internal component 316 to circuit board 331.For example, layer 329 may be a double-sided adhesive tape which may behydrophilic to support the capillary flow of fluid.

Analysis channel 330 may be defined by a wall(s) of internal component316, a wall(s) of layer 329, and/or a wall(s) of circuit board component331. For example, the top wall of analysis channel 330 may be defined byinternal component 315, the side walls of analysis channel 330 may bedefined by layer 329, and the bottom wall of analysis channel 330 may bedefined by circuit board 331. Additionally, each reservoir 317, 318, 319includes an outlet which connects the reservoir to analysis channel 330.In this manner, fluid within each of the reservoirs can flow throughtheir respective outlets and into analysis channel 330. Analysis channel330 may extend from the reservoirs to absorbent pad 328. Preferably, oneor more sensors on circuit board 331 are at least partially positionedwithin analysis channel 330.

Circuit board 331 is disposed within housing 304 and may be coupled tointernal component 316, e.g., via layer 329. Circuit board 331 includeselectrical components, for example, one or more of: resistors,electrical leads, vias, and sensors needed for detection of targetanalytes. Although described separately, it is to be appreciated thatelectrical components of circuit board 331 need not be separatestructural elements. One or more electrical components and/or circuitsmay perform some of or all the roles of the various components describedherein.

Memory 332 is disposed within housing 304 and electrically coupled tocircuit board 331. Memory 332 may be any type of memory suitable forstoring data related to cartridge device 300 such as an EPROM, EEPROM,flash memory, or the like. Memory 332 may store data such as informationon cartridge type (e.g., inflammation, influenza, testosterone,fertility, Vitamin D), cartridge identification information (e.g.,serial number), and/or calibration information. When cartridge device300 is electrically coupled to reader device 400, reader device 400 mayreceive data transmitted from memory 332 to facilitate determination ofthe presence, absence, and/or quantity of one more target analytes usingsuch data. In one embodiment, one or more cartridge devices of a selectgroup of cartridges (e.g., common lot of production cartridges) may betested using a known quantity of target analytes to determine electricalproperties associated with one or more target analytes sensed by thesensor of the tested devices. Calibration information based on the testresults may be stored in memory 332 in the select group of cartridges toprecisely and consistently determine the presence, absence, and/orquantity of one more target analytes using the electrical signalsgenerated by the sensor of the cartridge and the calibrationinformation. Memory 332 also may store test result reliabilityinformation such as a predetermined range(s) of parameter(s), e.g.,voltage, current, that may be compared to electrical signals generatedby a positive control working electrode to determine whether theparameter(s) are within the predetermined range(s), as described below.

Referring now to FIGS. 6A, 6B, and 6C, exemplary circuit board and layerare illustrated, wherein FIG. 6A depicts circuit board 331, FIG. 6Bdepicts layer 329, and FIG. 6C depicts layer 329 disposed on and coupledto circuit board 331.

As shown in FIG. 6A, circuit board 331 may include heating elements 333,334, 335, 336, and/or 337, and sensor 338 which may include referenceelectrode 339, working electrode 340, counter electrode 341, backgroundworking electrode 342, and/or reference electrode 343. Working electrode340 may be masked with one or more striations 344 and background workingelectrode 342 may be masked with one or more striations 345. Circuitboard 331 may further include contacts 346 and 347 for electricallycoupling circuit board 331 to sonicator element 327 via wires, althoughsonicator element 327 also may be electrically coupled to circuit board331 with a spring contact as described below.

Heating elements 333, 334, 335, 336, and 337 are configured to generateheat within housing 304, e.g., based on electric signals transmittedfrom reader 400 at times specified in a protocol stored within thememory of reader 400. Each of heating elements 333, 334, 335, 336, and337 may form part of circuit board 331. For example, heating elements333, 334, 335, 336, and 337 may be a resistive heating element appearingas a serpentine trace located on the bottom side of circuit board 331,surrounding a via. In other embodiments, the heating element is locatedexternal to the cartridge, for example, on the reader. In variousembodiments in which a resistive heating element is used, in order togenerate heat, current is allowed to flow through the resistive heatingelement, for example, through actuation of a transistor. Current passingthrough the resistive heating element generates heat through Jouleheating. The heat is conducted to the via due to physical contactbetween the resistive heating element and the via. Heating elements 333,334, 335, 336, and 337 may be masked, e.g., with a solder mask, tomaintain heat transfer while promoting electrical isolation from sensor338 to minimize interference with electrical signals sensed by sensor338.

Heating element 333 may be positioned adjacent to an outlet of samplepreparation reservoir 317. The outlet may have a phase-changeablematerial therein to occlude the entire cross-section of the outlet,thereby fluidicly sealing the outlet. Heating element 333 may beconfigured to heat the phase-changeable material within the outlet ofsample preparation reservoir 317 such that the phase-changeable materialunseals the outlet of sample preparation reservoir 317 to permit thefluid having the sample mixed therein held in sample preparationreservoir 317 to flow into analysis channel 330. Heating element 333 maybe caused to heat the phase-changeable material at a time specified by aprotocol stored in the memory of reader 400, e.g., after reader 400detects cartridge device 300 electrically coupled thereto and afterreader 400 detects proper insertion of sample collection device 200 intocartridge device 300.

Heating element 334 may be positioned adjacent to an outlet of washreservoir 318. The outlet may have a phase-changeable material thereinto occlude the entire cross-section of the outlet, thereby fluidiclysealing the outlet. Heating element 334 may be configured to heat thephase-changeable material within the outlet of wash reservoir 318 suchthat the phase-changeable material unseals the outlet of wash reservoir318 to permit the wash solution held in wash reservoir 318 to flow intoanalysis channel 330. Heating element 334 may be caused to heat thephase-changeable material at a time specified by the protocol stored inthe memory of reader 400, e.g., a predetermined time after reader 400causes heating element 333 to be heated and/or a predetermined timeafter reader 400 causes heating element 336 to be heated.

Heating element 335 may be positioned adjacent to an outlet of substratereservoir 319. The outlet may have a phase-changeable material thereinto occlude the entire cross-section of the outlet, thereby fluidiclysealing the outlet. Heating element 335 may be configured to heat thephase-changeable material within the outlet of substrate reservoir 319such that the phase-changeable material unseals the outlet of substratereservoir 319 to permit the fluid with the substrates held in substratereservoir 319 to flow into analysis channel 330. Heating element 335 maybe caused to heat the phase-changeable material at a time specified bythe protocol stored in the memory of reader 400, e.g., a predeterminedtime after reader 400 causes heating element 334 to be heated.

Heating element 336 may be positioned adjacent to a fluidic isolatorwhich may comprise a phase-changeable material. Heating element 336 maybe configured to heat the phase-changeable material of the fluidicisolator after the outlet of sample preparation reservoir 317 isunsealed such that the phase-changeable material of the fluidic isolatorflows into analysis channel 330 to fluidicly isolate sample preparationreservoir 317 from substrate reservoir 319. Heating element 336 may becaused to heat the phase-changeable material at a time specified by theprotocol stored in the memory of reader 400, e.g., a predetermined timeafter reader 400 causes heating element 333 to be heated.

Heating element 337 may be positioned adjacent to a pocket of gas, e.g.,air, within analysis channel 330. Heating element 337 may be configuredto heat the pocket of air to cause the air to expand and put pressure onthe phase-changeable material, thereby facilitating movement of thephase-changeable material in analysis channel 330. Heating element 336may be caused to heat the phase-changeable material at a time specifiedby the protocol stored in the memory of reader 400, e.g., apredetermined time after reader 400 causes heating element 333 to beheated. Placement of heating element 337 in the downstream direction ofheating elements 333, 334, and 335 is expected to reduce bubbleformation in analysis channel 330.

Electrical leads (shown in FIG. 8) of circuit board 331 may be providedto establish electrical connections and continuity with a reader device.The electrical leads may be electrically coupled to heating elements333, 334, 335, 336,337, sensor 338 including each of reference electrode339, working electrode 340, counter electrode 341, background workingelectrode 342, and reference electrode 343, contacts 346, 347, and tomemory 332. In this manner, such components may receive electricalcurrent when activated by the reader device. Advantageously, while theelectrical leads are exposed at the electrical connector portion on thebottom surface of circuit board 331 (shown in FIG. 4B), the electricalleads electrically coupling the connectors to the components may betraceless on the top surface of circuit board 331 as shown in FIG. 6A.The traceless configuration of circuit board 331 between the electricalconnector portion and these components creates a smooth top surface ofcircuit board 331 to reduce bonding interferences, thereby promotingsecure adhesion, with layer 329. The bonding interferences may causeleakage when fluid enters analysis channel 330 due to malformation oflayer 329 caused by such interferences.

Heating elements 333, 334, 335, 336, 337 may be formed of a conductorand each may include a via. A via is a standard product on printedcircuit boards and is typically used to enable signal traces on onelayer of a circuit board to continue electrically with another layer.The vias provide electrical continuity through multiple layers. Suchvias are excellent conductors of heat; they are able to transfer heat toa very precise location without affecting the surrounding areas, becausethe surrounding material that comprises most circuit boards is anexcellent insulator of heat. Thus, in various embodiments, a pluralityof vias are provided in circuit board 331 as heating elements, and eachvia is disposed under, over, or adjacent to a phase-changeable,heat-actuated valve disposed in a reservoir outlet to create a valveactuating element. The precision of heat transfer associated with thevias allows for minimal crosstalk between valves located close to eachother; thus, the timing of valve actuation can be carefully controlledfor each valve. The valves may be formed of a phase-changeable materialsuch as wax, for example, a hydrophilic wax, and the vias act asconductors of heat to melt wax at precise points of time, as controlledby a reader device. Upon phase transition, e.g., melting, of a wax valvedisposed in the outlet of a reservoir, the outlet is no longer occludedand the reservoir has an opening through which its fluid contents candrain into the analysis channel. The holes in the vias may be filledwith a filling material, e.g., solder, and the vias may be masked, e.g.,with a solder mask, to maintain heat transfer while promoting electricalisolation from sensor 338 to minimize interference with electricalsignals sensed by sensor 338.

In order to ensure full melting of the wax with precise timing, invarious embodiments, the wax valves are carefully constructed within theoutlets of the reservoirs. For example, in some embodiments, it ispreferable for the wax valves to have the minimum height necessary toocclude the outlet of the reservoir; the minimal height minimizes thedistance heat must travel to melt the wax. One example method forrealizing a wax barrier having such characteristics involves applyingmelted wax to a pre-heated via. Advantageously, when the via ispre-heated, it takes longer for the wax valve to solidify relative to aroom-temperature via; thus the wax has more time to flatten and expandoutward before hardening. “Pancaking” of the wax is desirable tominimize the height, which will maximize the chance of proper meltingactuation of the valve. Additionally, the heating of the via facilitatesa greater level of contact area between the wax and the via such that agreater proportion of the wax experiences the heat, also maximizing thechance of proper valve actuation. The method of heating the via prior todeposition of wax is further enhanced with the following method: theopening of the reservoir is aligned over the via such that when themelted wax is applied to the pre-heated via, the opening at the bottomof the reservoir is spatially close to the via such that when the waxhardens, the wax adheres simultaneously to multiple inner walls of thereservoir and the via itself. This is advantageous for enhancing themanufacturing yield of intact valves that fully occlude the opening tothe analysis channel such that no inadvertent flow of fluid from thereservoir occurs.

Sensor 338 may be configured to be exposed to the fluid in analysischannel 330 and to generate a signal indicative of the presence,absence, and/or quantity of one or more analytes within the sample.Sensor 338 may detect electrical signals resulting from chemicalreactions over sensor 338. For example, the mixed fluid from samplepreparation reservoir 317 may be introduced into analysis channel 330and signaling agents in the mixed fluid may localize over sensor 338(e.g., responsive to magnetic fields holding magnetic particles (ifpresent) directly or indirectly bound to the signaling agents). Thechemical reactions may occur when fluid from substrate reservoir 319reacts with the particles from mixed fluid from sample preparationreservoir 317 localized over sensor 338. For example, a substratesolution having a substrate may be introduced from substrate reservoir319 and sensor 338 may detect electrical signals resulting from thereactions between the substrate (e.g., TMB, OPD) and the signalingagents (e.g., HRP, SBP) localized over sensor 338. The reactions maycause electrons to be stripped from the substrate by the signalingagents (which electrons may be donated to acceptor molecules from thesubstrate solution) thereby generating electrical signals detectable bysensor 338. Such detected electrical signals may be used to generate thesignal indicative of the presence, absence, and/or quantity of one ormore analytes within the sample. The signal may be transmitted to readerdevice 400, e.g., via respective electrical connectors of cartridgedevice 300 and reader device 400.

Sensor 338 may include reference electrode 339, working electrode 340,counter electrode 341, background working electrode 342, and/orreference electrode 343. Sensor 338 is disposed in analysis channel 330and the area of analysis channel 330 above sensor 338 may be referred toas the “analysis zone.” Sensor 338 is strategically located such that,when circuit board 331 is included within the assembled cartridge 300with a surface of circuit board 331 forming one wall of analysis channel330, sensor 338 is at least partially disposed within analysis channel330. While one sensor is illustrated, a plurality of sensors may beprovided, each spaced relative to the others, and preferably all alignedwithin analysis channel 330. In addition, working electrode 340 andbackground working electrode 342 may be disposed upstream and downstreamof one another, or vice versa, and sensor 338 may include additionalworking electrodes beyond working electrode 340 and background workingelectrode 342.

Sensor 338 may be an electrochemical sensor that forms anelectrochemical cell within analysis channel 330. Reference electrode339 may be configured to create a voltage differential between itselfand working electrode 340. Counter electrode 341 may provide electrons(e.g., from the substrate stripped by the signaling agents) which gatheron working electrode 340 when the electrical environment created byreference electrode 339 and working electrode 340 results in a positivecharge over working electrode 340. As is explained above, an oxidationreaction may occur at sensor 338 if an oxidizing enzyme (e.g., asignaling agent described herein such as HRP, SBP which may beintroduced into analysis channel 330 from sample preparation reservoir317) bound indirectly to a particle (e.g., a magnetic particle which maybe introduced into analysis channel 330 from sample preparationreservoir 317) is present at sensor 338 and an appropriate chemicalsubstrate (e.g., TMB, OPD) is introduced into analysis channel 330(e.g., from substrate reservoir 319). In such embodiments, workingelectrode 340 releases electrons to replenish electrons stripped fromthe substrate by the oxidizing enzyme in a quantity proportional to theamount of oxidizing enzyme present. The release of electrons fromworking electrode 340 (e.g., from the substrate reacting with thesignaling agent over working electrode 340) is a current which may bedetectable as a signal within a circuit connected to sensor 338. Sensor338 can thereby indirectly detect the presence, absence, and/or quantityof oxidizing enzymes localized in the analysis zone. A processor, forexample, within the reader device described below, can then correlatethe presence, absence, and/or quantity of one or more target analytes tothe presence, absence, and/or quantity of oxidizing enzymes. Thefunctions of such a processor are described in more detail below. One ormore magnetic fields may be used to facilitate localization of theenzymes or other signaling agents within the analysis zone.Advantageously, in such embodiments, no affinity molecules need to bepre-bound to sensor 338 to achieve localization, which would otherwisesignificantly slow the analyte quantification process due to the limitsof diffusion-based hybridization kinetics. Details of the magneticfields are also provided below.

Sensor 338 may include gold surfaces made through an ENIG process. Inother embodiments, gold or gold-plated sensors are used that have notbeen made through an ENIG process. A person skilled in the art canappreciate that there are many plating processes for catalytic andautocatalytic deposition of gold utilized to create electrically activepads within the printed circuit board industry. Working electrode 340may have a surface chemistry formed of a self-assembled monolayer suchas thiolated ethylene glycol and/or a dithiol such as hexaethyleneglycol dithiol for added stability. The hydrophilic nature of the headgroups of such surface chemistry facilitates flow and proteinresistance. Additionally or alternatively, the surface of one or more ofthe electrodes may be backfilled with mercaptoundecanoic acid,mercaptohexanol, or other suitable backfiller. The surface of one ormore of the electrodes within sensor 338 may be formed throughsequential addition and incubation of the ethylene glycol dithiol andthe backfiller at unelevated temperatures.

Background working electrode 342 may be ambient electrochemical noisesensors spaced within analysis channel 330 away from the site ofmagnetic particle localization. Background working electrode 342 may beused to quantify background noise downstream or upstream of workingelectrode 340, depending on the selected order of working electrode 340and background working electrode 342 within analysis channel 330. Suchnoise may be due to, for example, the presence of non-specifically boundenzyme. During processing of the detection results, a processor atreader 400 may apply an algorithm to remove the background workingelectrode signal(s) (from background working electrode 342) from thedetection sensor signal (from working electrode 340) to account forand/or eliminate system noise and to thereby allow for properquantification or detection of the one or more target analytes. Thesignal from background working electrode 342 may be used for errordetection and diagnosis of an improperly functioning cartridge, e.g., asevidenced by the signal from background working electrode 342 havingelectrical value(s) outside a predetermined range(s).

Reference electrode 343 may be configured to create a voltagedifferential between itself and background working electrode 342.Counter electrode 341 also may provide electrons which gather onbackground working electrode 342 when the electrical environment createdby reference electrode 343 and background working electrode 342 resultsin a positive charge over background working electrode 342.

In some embodiments, the detection is carried out using a standardelectrochemical circuit that utilizes a bias potential generated atbackground working electrode 342 for the oxidation/reduction reaction toproceed. The potential is held at the reduction potential of thechemical substrate (low enough that there is little nonspecificreduction of reducible species in the solution) so that the flow ofelectrons to the oxidized molecules can be quantified using anoperational amplifier based current-to-voltage (op amp) circuit topologyin reader device 400 electrically connected to working electrode 340.

A common substrate molecule, tetramethylbenzidine, may be used for HRP.When present, HRP oxidizes TMB molecules, and these molecules are inturn reduced by working electrode 340. Since this event occurs inproportion to the amount of HRP present which in turn is proportional tothe amount of target analyte present, a change in the current-to-voltageop amp measurement results. Using an analog-to-digital converter, theactual signal can be delivered to a processor for processing. Asdescribed in more detail below, in various embodiments, the processorand signal processing components are provided within the reader device.

Working electrode 340 may be masked, e.g., solder masked, with aplurality of striations 344 configured to promote homogenousdistribution and retention of the plurality of magnetic particlesreleased from sample preparation reservoir 317 over working electrode340. Accuracy of analyte detection may be adversely impacted bypremature wash away of magnetic particles during analysis due, forexample, force on the particles in the analysis channel flow directioncaused by release of the wash solution from wash reservoir 318 and/orthe fluid having the chemical substrates from substrate reservoir 319being greater than the magnetic force toward the magnetic generators ofreader 400 disposed beneath working electrode 340 during analysis. Suchstriations 344 promote resistance to movement of the plurality ofmagnetic particles off working electrode 340. In addition, backgroundworking electrode 342 may be masked, e.g., solder masked, with aplurality of striations 345, although such striations are not necessaryon background working electrode 342 and are merely exemplary of analternate embodiment.

In alternative embodiments, sensor 338 may be configured to analyze thefluid in analysis channel 330 and to generate a signal indicative of thepresence, absence, and/or quantity of one or more analytes within thesample, wherein the signal is visible. For example, the housing of thecartridge device may include a window that permits a user to view, e.g.,with a camera, fluorescence and quantify that fluorescence to determinethe presence, absence, and/or quantity of one or more analytes withinthe sample.

Referring now to FIG. 6C, layer 329 is disposed on the top surface ofcircuit board 331 such that sensor 338 is disposed at least partially inanalysis channel 330. Heating elements 333, 334, 335, 336, and 337 maybe covered, partially or fully, with masks 348, 349, 350, 351, and 352,respectively. Masks 348, 349, 350, 351, and 352 may be solder masks.Mask 348 is configured to maintain heat transfer from heating element333 to the phase-changeable material within the outlet of samplepreparation reservoir 317 at an energy level sufficient to cause a phasechange of the material while promoting electrical isolation betweenheating element 333 and sensor 338 to minimize interference by heatingelement 333 with the electrical signals sensed by sensor 338. Mask 349is configured to maintain heat transfer from heating element 334 to thephase-changeable material within the outlet of wash reservoir 318 at anenergy level sufficient to cause a phase change of the material whilepromoting electrical isolation between heating element 334 and sensor338 to minimize interference by heating element 334 with the electricalsignals sensed by sensor 338. Mask 350 is configured to maintain heattransfer from heating element 335 to the phase-changeable materialwithin the outlet of substrate reservoir 319 at an energy levelsufficient to cause a phase change of the material while promotingelectrical isolation between heating element 335 and sensor 338 tominimize interference by heating element 335 with the electrical signalssensed by sensor 338. Mask 351 is configured to maintain heat transferfrom heating element 336 to the phase-changeable material of the fluidicisolator at an energy level sufficient to cause a phase change of thematerial while promoting electrical isolation between heating element336 and sensor 338 to minimize interference by heating element 336 withthe electrical signals sensed by sensor 338. Mask 352 is configured tomaintain heat transfer from heating element 337 to the pocket of gas inanalysis channel 330 above heating element 337 at an energy levelsufficient to cause movement downstream in analysis channel 330 ofphase-changeable material while promoting electrical isolation betweenheating element 337 and sensor 338 to minimize interference by heatingelement 337 with the electrical signals sensed by sensor 338.

Referring now to FIGS. 7A, 7B, and 7C, circuit board 331′ and layer 329′are constructed similarly to circuit board 331 and layer 329 of FIGS.6A, 6B, and 6C except that heating elements 333′, 334′, 335′, 336′, and337′ are positioned in a different configuration on circuit board 331′and analysis channel 330′ is re-shaped accordingly. In addition, FIG. 7Cdepicts absorbent pad 328 coupled to layer 329′ at the downstream end ofanalysis channel 330′.

Referring now to FIGS. 7D and 7E, alternative exemplary sensors that maybe used in the cartridges described herein are provided. Sensor 338″ mayinclude reference electrode 339″, working electrode 340″, counterelectrode 341″, negative control working electrode 342″ (also referredto herein as a background working electrode), and/or positive controlworking electrode 376. Sensor 338″ may detect electrical signalsgenerated by chemical reactions at sensor 338″ as described above forsensor 338. Sensor 338″ is disposed in the analysis channel in the samemanner as sensor 338 described above. While one sensor is illustrated, aplurality of sensors may be provided, each spaced relative to theothers, and preferably all aligned within the analysis channel.Preferably, fluid flows from the reservoirs in the analysis channel andtravels over the electrodes in the following order: positive controlworking electrode 376, reference electrode 339″, counter electrode 341″,working electrode 340″, and negative control working electrode 342″.

Sensor 338″ may be an electrochemical sensor that forms anelectrochemical cell within the analysis channel. Positive controlworking electrode 376 may have affinity molecules pre-bound to thesurface of positive control working electrode 376 to achievelocalization of oxidizing enzymes, or other signaling agents, overpositive control working electrode 376. The affinity molecules may besurface bound antibodies. Positive control working electrode 376 may beconfigured to detect current generated by reactions between an oxidizingenzyme, or other signaling agents, indirectly bound to the affinitymolecules and an appropriate chemical substrate introduced into theanalysis channel, e.g., from the substrate reservoir. In suchembodiments, positive control working electrode 376 releases electronsto replenish electrons stripped from the substrate by the oxidizingenzyme in a quantity proportional to the amount of oxidizing enzymepresent. The release of electrons from positive control workingelectrode 376 is a current which may be detectable as a signal within acircuit connected to sensor 338″. A processor, for example, within thereader device described below, may process the signal to determine ifthe signal indicates a quantity of oxidizing enzyme, or other signalingagent, within a predetermined range which may be stored in memory of thecartridge and/or reader device. If the detected quantity is within therange, the processor may verify the cartridge and continue processingsignals to determine the presence, absence, and/or quantity of one ormore target analytes within the sample. The signal from positive controlworking electrode 376 may be used for error detection and diagnosis ofan improperly functioning cartridge, e.g., as evidenced by the signalfrom positive control working electrode 376 having electrical value(s)outside a predetermined range(s). For example, the processor of thereader may generate an error alert if the signal from the positiveworking control electrode 376 is outside a predetermined range and/ormay consider a reading from working electrode 340″ acceptable if withinthe predetermined range.

Reference electrode 339″ may be configured to create a voltagedifferential between itself and working electrode 340″. Counterelectrode 341″ may provide electrons which gather on working electrode340″ when the electrical environment created by reference electrode 339″and working electrode 340″ results in a positive charge over workingelectrode 340″. Reference electrode 339 and/or counter electrode 341″may have a surface chemistry formed of a self-assembled monolayer suchas thiolated ethylene glycol and/or a dithiol such as hexaethyleneglycol dithiol for added stability. The hydrophilic nature of the headgroups of such surface chemistry facilitates flow and proteinresistance. Additionally or alternatively, the surface of referenceelectrode 339 and/or counter electrode 341″ may be backfilled withmercaptoundecanoic acid, mercaptohexanol, or other suitable backfiller.

Sensor 338″ may be used when magnetic particles are not present in thecartridge. For example, working electrode 340″ may have affinitymolecules pre-bound to the surface of working electrode 340″ to achievelocalization of oxidizing enzymes, or other signaling agents, overworking electrode 340″. The affinity molecules may be surface boundantibodies. Working electrode 340″ may be configured to detect currentgenerated by reactions between an oxidizing enzyme, or other signalingagents, indirectly bound to the affinity molecules and an appropriatechemical substrate introduced into the analysis channel, e.g., from thesubstrate reservoir. In such embodiments, working electrode 340″releases electrons to replenish electrons stripped from the substrate bythe oxidizing enzyme in a quantity proportional to the amount ofoxidizing enzyme present. The release of electrons from workingelectrode 340″ is a current which may be detectable as a signal within acircuit connected to sensor 338″. Sensor 338 can thereby indirectlydetect the presence, absence, and/or quantity of oxidizing enzymeslocalized in the analysis zone. A processor, for example, within thereader device described below, can then correlate the presence, absence,and/or quantity of one or more target analytes to the presence, absence,and/or quantity of oxidizing enzymes. The functions of such a processorare described in more detail below.

Working electrode 340″ illustratively does not include a plurality ofstriations as sensor 338″ may be used to detect the presence, absence,and/or quantity of one or more target analytes without the use ofmagnetic particles.

Negative control working electrode 342″ may be ambient electrochemicalnoise sensors spaced within the analysis channel away from the site oflocalization. Negative control working electrode 342″ may be used toquantify background noise downstream of working electrode 340″. Suchnoise may be due to, for example, the presence of non-specifically boundenzyme. During processing of the detection results, a processor atreader 400 may apply an algorithm to remove the negative control workingelectrode signal(s) (from negative control working electrode 342″) fromthe detection sensor signal (from working electrode 340″) to account forand/or eliminate system noise and to thereby allow for properquantification or detection of the one or more target analytes. Thesignal from negative control working electrode 342″ may be used forerror detection and diagnosis of an improperly functioning cartridge,e.g., as evidenced by the signal from negative control working electrode342″ having electrical value(s) outside a predetermined range(s). Forexample, the processor of the reader may generate an error alert if thesignal from the negative working control electrode 342″ is above athreshold and/or may consider a reading from working electrode 340″acceptable if below the threshold.

Negative control working electrode 342″ may have a surface chemistryformed of a self-assembled monolayer such as thiolated ethylene glycoland/or a dithiol such as hexaethylene glycol dithiol for addedstability. The hydrophilic nature of the head groups of such surfacechemistry facilitates flow and protein resistance. Additionally oralternatively, the surface of Negative control working electrode 342″may be backfilled with mercaptoundecanoic acid, mercaptohexanol, orother suitable backfiller.

Referring now to FIG. 7E, sensor 338′″ may be used when magneticparticles are present in the cartridge. Like sensor 338″, sensor 338′″includes positive control working electrode 376′, reference electrode339′″, counter electrode 341′″, and negative control working electrode342′″ structured similarly to the like-primed components of FIG. 7Ddescribed above. Working electrode 340′″ may be structurally similar toworking electrode 340 described above with respect to FIG. 6A. In thismanner, sensor 338′″ is particularly well suited for target analytedetection using one or more magnetic fields to facilitate localizationof the enzymes or other signaling agents within the analysis zone.

Referring now to FIG. 7F, an alternative exemplary sensor that may beused in the cartridges described herein is provided. Sensor 338″″ may bestructured the same manner as sensor 338′″ except that negative controlworking electrode 342″″ may be positioned between positive controlworking electrode 376″ and reference electrode 339″″. Preferably, fluidflows from the reservoirs in the analysis channel and travels over theelectrodes in the following order: positive control working electrode376″, negative control working electrode 342″″, reference electrode339″″, counter electrode 341″″, and working electrode 340″″.

Referring to FIG. 8A, internal component 316 is coupled to circuit board331, e.g., via layer 329 positioned therebetween. Internal component 316may include sample preparation reservoir 317 having outlet 353 withvalve 354 positioned adjacent to heating element 333, wash reservoir 318having outlet 355 with valve 356 positioned adjacent to heating element334, and substrate reservoir 319 having outlet 357 with valve 358positioned adjacent to heating element 335. Each of reservoirs 317, 318,and 319 is, at least at times, in fluid communication with analysischannel 330 such that fluid exiting the reservoirs, e.g., via theirrespective outlets when the respective valves are opened, flows intoanalysis channel 330. In addition, fluidic isolator 359 may bepositioned adjacent to heating element 336 and in fluidic communicationwith analysis channel 330.

Valves 354, 356, and 358 may be located within outlets 353, 355, and357, respectively, at the bottom of reservoirs 317, 318, and 319,respectively, of cartridge 300. Outlets 353, 355, and 357 may each beformed of a hole within a bottom wall of internal component 316 aboveanalysis channel 330. Valves 354, 356, and 358 may each be formed of aheat-sensitive, phase-changeable material, such as, for example, ahydrophilic wax. Prior to actuation, the wax or other heat-sensitivematerial of the valve is in a solid or semi-solid state and is sized andshaped to fill an entire cross-section of the outlet such that no fluidcan escape from the respective reservoir into analysis channel 330.Valves 354, 356, and 358 may be aligned directly above one or moreheating elements (with a solder mask therebetween) or other localizedheat-conductive element. Such alignment allows for the localizedapplication of heat to induce a phase change in the valve withoutcausing a phase change of any neighboring valves. In variousembodiments, the phase change melts or otherwise transforms theheat-sensitive material such that it no longer causes full occlusion ofthe outlet, but instead permits fluid in the respective reservoir toflow into analysis channel 330.

In addition, fluidic isolator 359 may be formed of a heat-sensitive,phase-changeable material, such as, for example, a hydrophilic wax.Prior to actuation, the wax or other heat-sensitive material is in asolid or semi-solid state and is disposed out of the flow path betweenthe outlets of the respective reservoirs and sensor 338 within analysischannel 330. Fluidic isolator 359 may be sized and shaped to block theflow path on analysis channel 330 between an outlet of one reservoir,e.g., sample preparation reservoir 317, and the outlet of anotherreservoir, e.g., substrate reservoir 319, when fluidic isolator isactivated, e.g., by heating heating element 336. Fluidic isolator 359may be aligned directly above heating element 336 (with a solder masktherebetween) or other localized heat-conductive element. Such alignmentallows for the localized application of heat to induce a phase change influidic isolator 359 without causing a phase change of any neighboringvalves. In various embodiments, the phase change melts or otherwisetransforms the heat-sensitive material such that it flows into analysischannel to block outlet 353 of sample preparation reservoir 317 fromanalysis channel 330. In this manner, fluid from substrate reservoir319, when released into analysis channel 330, cannot enter into samplepreparation reservoir 317 and cannot interact with leftover signalingagents from sample preparation reservoir 317.

The wax material disposed upon the via or a solder mask over via, andwhich occludes the opening of the respective reservoir or isolates theanalysis channel, may be a hydrophilic material such as hexadecanol oroctodecanol. This advantageously promotes, rather than obstructs theflow of fluid past any wax bits that harden within any area of theanalysis channel after actuation. These materials also preferably have amelting temperature between 50 and 100 degrees Celsius, which allows foractuation with reasonable power-consumption for a battery-operateddevice, yet remains unactuated in general handling and storageenvironments and/or during a sonication protocol. The amount of wax pervalve may be below 1 microliter in its liquid state, the amount may beless than or equal to 0.5 microliters, and the amount may be greaterthan 2 nanoliters. Using minimal amount of wax in the valves is one wayto reduce any occlusion of the analysis channel and maximize full valveactuation when heat is applied. The valve also may have afeedback-and-control system that allows for a consistent thermal profileto be achieved at the via for consistent valve actuation. Furthermore,this feedback-and-control system may incorporate sensing elements toenable the system to confirm that each valve has properly actuated.

As shown in FIG. 8A, circuit board 331 may have exposed leads 360 atelectrical connector 312. In one embodiment, leads 360 are exposed onlyon the bottom surface of circuit board 331 although leads 360 may beexposed on the top surface of circuit board 331 but are preferablytraceless as shown. As described above, electrical connector 312 permitselectrical connection with a corresponding electrical connector ofreader device 400 such that cartridge device 300 may transmit signalsindicative of the presence, absence, and/or quantity of one or moretarget analytes within a collected sample sensed by the sensor ofcartridge device 300.

Internal component 316 may include absorber pad housing 361 sized andshaped to hold absorber pad 328. Absorber pad housing 361 may include aplurality of vent holes 362 to permit exposure of absorber pad 328within absorber pad housing 361 to the environment within housing 304 ofcartridge device 300.

Input tunnel 301 of cartridge device 300 may include slot 363 configuredto permit seal piercer 321 to be at least partially disposed withininput tunnel 301. Slot 363 may be at the proximal end of input tunnelcomponent 326 as illustrated. Slot 363 may be sized and shaped to permitengager 324 of slider 321 to be disposed within input tunnel 301. Inaddition, slot 363 may have a length sufficient to permit slider 322 toslide, when engager 324 contacts the sample collection device withininput tunnel 301, distally from a pre-venting position to a ventingposition.

Referring now to FIGS. 8B and 8C, a close-up view of certain componentsof circuit board 331 and a valve are shown. FIG. 8B depicts conductor364 coupled to resistor 365, e.g., an aluminum resistor, which iscoupled to heating element 333 and FIG. 8C further shows mask 348disposed between heating element 333 and valve 354. As will be clear toone skilled in the art, while the details of heating element 333, mask348, and valve 354 are illustrated, such a configuration may be utilizedwith respect to heating elements 334, 335, 336, and 337 with theirrespective masks and valves, fluidic isolators, or air pockets. Currentfrom reader device 400 may pass from conductor 364 through resistor 365to generate heat through Joule heating. The heat is conducted to heatingelement 333 due to physical contact between resistor 365 and heatingelement 333. Heating element 333 generates heat through mask 348 tocause a phase change of the phase-changeable material of valve 354 whilepromoting electrical isolation from sensor 338 to minimize interferenceby heating element 333 with electrical signals sensed by sensor 338.

Referring now to FIG. 8D, a close-up view of alternative components ofcircuit board 331′ and a valve are shown. FIG. 8D depicts conductors364′, e.g., solder pads, coupled to resistor 365′, e.g., an aluminumresistor. Unlike the configuration shown in FIGS. 8B and 8C, conductor364′ is not coupled to heating element 333′ (which includes a via incircuit board 331′) such that a mask disposed between heating element333′ and valve 354′ is not needed. As will be clear to one skilled inthe art, while the details of heating element 333′ and valve 354′ areillustrated, such a configuration may be utilized with respect toheating elements 334, 335, 336, and 337 with their respective valves,fluidic isolators, or air pockets. Current from reader device 400 maypass from conductor 364′ through resistor 365′ to generate heat throughJoule heating. The via of heating element 333′ is disposed between, andelectrically isolated from, conductors 364′ coupled to resistor 365′.The heat is conducted to heating element 333′ through indirect contactbetween resistor 365′ and heating element 333′. Heating element 333′generates heat to cause a phase change of the phase-changeable materialof valve 354′ while promoting electrical isolation from the sensor tominimize interference by heating element 333′ with electrical signalssensed by the sensor.

FIGS. 9A and 9B illustrate various shuttles that may be disposed withinthe input tunnel of the cartridge device. Referring to FIG. 9A, shuttle324 may include first end 366, reagent ball compartment 367, compartmentdivider 368 having slot 369, sample compartment 370, second end 371having opening 372 therethrough, and/or beams 373, 374.

Shuttle 324 is configured to be disposed within the cartridge housing,preferably within the input tunnel between the sample preparationreservoir and the aperture defining the opening of the input tunnel in apre-mixing state. In such a pre-mixing state, first end 366 may form awall of the sample preparation reservoir to seal fluid within thereservoir. First end 366 may include one or more sealing members, e.g.,O-rings, to enhance a liquid tight seal that may be comprised in part ofchlorobutyl. The area between first end 366 and compartment divider 368may be referred to as reagent ball compartment 367. Reagent ballcompartment 367 is configured to house one or more reagent balls, e.g.,reagent ball 375. In the pre-mixing state, reagent ball compartment 367is preferably sealed off from the fluid in the sample preparationreservoir. Compartment divider 368 may be used to divide compartments inshuttle 324, e.g., reagent ball compartment 367 and sample compartment370. Compartment divider 368 may include slot 369 which permits fluid toflow through compartment divider 368 when compartment divider 368 isdisposed within the fluid of the sample preparation reservoir in anmixing state. Permitting fluid to flow through slot 369 may enhancemixing of the sample, the reagent ball, and the fluid in the samplepreparation reservoir. In addition, slot 369 permits enhanced fluidiccommunication between reagent ball 375 and desiccant 325 disposed withinthe cartridge housing in the pre-mixing state. It should be understoodwithout being explicitly stated that the compartment may contain reagentballs that are the same or different from each other, and can bepreselected for detection and/or quantification of the target analyte.

In the pre-mixing state, second end 371 may be disposed between firstend 366 and the aperture defining the opening of the input tunnel.Second end 371 may include opening 372 which is configured to permit thecollected sample to be inserted therethrough and into sample compartment370. For example, opening 372 may be sized slightly larger than the tipof the sample collection device such that the tip can travel throughopening 372 while opening 372 wipes excess sample from the tip. In thismanner, at most a predetermined volume of sample is inserted in samplecompartment 370. The area between second end 371 and compartment divider368 may be referred to as sample compartment 370. Shuttle 324 mayinclude one or more beams 373, 374 configured to couple first end 366 tosecond end 371 and to be coupled to compartment divider 368. Beams 373,374 are preferably positioned closer to the top surface of the cartridgehousing when in the input tunnel to avoid interference with fluid mixingin the mixing state.

Shuttle 324 may move from a first position in a pre-mixing state to asecond position in a mixing state, e.g., responsive to application of aforce beyond a threshold force exerted on shuttle 324 by the samplecollection device. In the mixing state, first end 366, reagent ballcompartment 367, and sample compartment 370 may be disposed within thesample preparation reservoir. Accordingly, the sample and the reagentball(s) may be mixed within fluid in the sample preparation reservoir.Second end 371 of shuttle 324 may re-form the wall of the samplepreparation reservoir to seal fluid therein when the sample and/or theone or more reagent ball are in the sample preparation reservoir.Preferably, the distal portion of the sample collection device fluidiclyseals opening 372 such that fluid cannot escape the sample preparationreservoir into the input tunnel in the mixing state. Second end 371 mayinclude one or more sealing members, e.g., O-rings, to enhance a liquidtight seal. In this manner, the sample preparation reservoir remainsfluidicly sealed in the pre-mixing state by first end 366 and in themixing state by second end 371 and the sample collection device andduring movement from the pre-mixing state to the mixing state. Inaddition, unlike a breakable membrane housing reagents, shuttle 324 mayremain intact as the sample is moved into sample preparation reservoir317 for analysis.

Reagent ball 375 may include one or more of magnetic particles, affinitymolecules, connection molecules, signaling agents, competitor bindingmolecules, competitor molecules, labels, signaling agents, primers,nucleic acid probes, and/or polymerases, and other enzymes or componentsas described in further detail herein and the components, encapsulationmaterial and dimensions may be the same or different from each other. Inone aspect, reagent ball 375 is formed by the freezing (i.e. loweringthe temperature of a volume of liquid (such as between 5 microliters and30 microliters)) to a temperature to induce a phase change in theliquid. The temperature may vary depending on the components of theliquid. In a further aspect, the liquid additionally comprisesexcipients known to those of ordinary skill in the art of lyophilizationsuch as lyoprotectants for the functional preservation of the reagentsthat may be temperature sensitive, e.g., nucleic acids and/or proteincomponents, within the liquid volume as it undergoes the process offreezing and drying to become reagent ball 375. Stabilizers such asdissacharides like sucrose and trehalose or other lyoprotectants such aspolethylene glycol of various molecular weights and bulking or cakingagents such as mannitol, glycine, povidone, and others known in the artcan comprise some of the final constituents of reagent ball 375 inaddition to the reagents noted herein. The (w/v) percentage of theexcipients within the volume of liquid to be freeze dried to formreagent ball 375 can vary widely from about 0.1% to about 30% and becombined in various ways, often with a combination of a dissacharide asa lyoprotectant and a caking agent or bulking agent to add structure.Reagent ball 375 can be of many sizes, non-limiting examples of suchinclude having an diameter of between about 1 mm to about 7 mm, oralternatively from about 2 mm to about 5 mm, or alternatively about 3mm, or alternatively less than about 7 mm, or alternatively less thanabout 5 mm, or alternatively less than about 4 mm. While reagent ball375 is illustrated as a sphere, the disclosure is not limited theretoand many shapes may be used and multiple reagent balls each containingthe same or different reagents also may be used. As is ordinary in theart, such liquid when formed containing the components and excipientscan be frozen through flash freezing in liquid nitrogen or through shelffreezing within a lyophilizer machine. After freezing, said frozenvolume is subjected potentially to annealing treatments forcrystalization of caking agents, primary drying and secondary dryingwherein water is removed until a sufficiently small percentage (e.g.<8%, preferably <5% and preferably around 1%) remains in the finalfreeze dried product, which is reagent ball 375.

Referring now to FIG. 9B, shuttle 324′ is constructed similarly toshuttle 324 of FIG. 9A except compartment divider 368′ is solid withouta slot and beams 377, 378, and 379 are positioned in differentorientations on shuttle 324′.

As will be readily apparent to one skilled in the art, while FIGS. 9Aand 9B illustrate shuttles having one sample compartment and one reagentball compartment, the disclosure is not limited thereto and the shuttlesmay define one or more compartments configured to receive the collectedsample from the sample collection device when inserted in the inputtunnel 301 and one or more additional compartments configured to houseone or more reagents balls.

Referring now to FIGS. 10A through 10P, insertion of a sample collectedby a sample collection device into a cartridge device is described.Prior to insertion within input tunnel 301 of cartridge device 300,sample collection device 200 is exposed to a sample, e.g., within aninner cheek, the throat, the mouth, a nasal passageway, an ear, fromurine, from blood, from plasma, from saliva, etc. Tip 204 of samplecollection device 200 is designed to retain some of the sample to permitanalysis of the presence, absence, and/or quantity of one or more targetanalytes within the sample using cartridge device 300 and reader device400. Cartridge device 300 may be electrically coupled to reader device400 before or after sample collection device 200 is inserted incartridge device 300.

Referring to FIG. 10A, the distal end of sample collection device 200 isfirst inserted into the aperture defining the opening of input tunnel301. Shuttle 324 is disposed within input tunnel 301 in a pre-mixingposition wherein first end 366 of shuttle 324 forms a wall of samplepreparation reservoir 317 to fluidicly seal fluid within samplepreparation reservoir 317. In this pre-mixing position, reagent ball 375housed by shuttle 324 is within input tunnel 301 and not exposed tofluid within sample preparation reservoir 317. As the distal end ofsample collection device 200 moves distally into input tunnel 301,sample collection device 200 may contact engager 380 of slider 322. Forexample, distal sealing zone 208 may contact engager 380 as illustrated.Distal sealing zone 208 may be angled to facilitate movement of engager380 past distal sealing zone 208 as sample collection device 200 ismoved more distally or distal sealing zone 208 may be a shoulder sizedto cause movement of slider 322.

As shown in FIG. 10B, tip 204 (having sample thereon and/or within tube205) enters sample compartment 370 of shuttle 324 through opening 372 ofsecond end 371. Preferably, as tip 204 enters sample compartment 370,shuttle 324 remains substantially in position within input tunnel 301and first end 366 of shuttle 324 continues to form a wall of samplepreparation reservoir 317 to seal fluid within sample preparationreservoir 317. In addition, first end 366 of shuttle 324 may continue toform a wall of sample preparation reservoir 317 to seal fluid withinsample preparation reservoir 317 (and shuttle 324 may remainsubstantially in pre-mixing position) until distal sealing portion 208contacts second end 371 to fluidicly seal opening 372. Application of aforce greater than a threshold force on shuttle 324 (e.g., at second end371) by sample collection device 200 (at distal sealing portion 208) maymove shuttle 324 from the pre-mixing position to the mixing positionwherein the sample on sample collection device 200 and reagent ball 375are mixed within fluid of sample preparation reservoir 317.

In addition, as sample collection device 200 (e.g., tip 204) is insertedinto shuttle 324 (e.g., via opening 372), shuttle 324 may wipe excesssample from sample collection device 200, thereby preventing the wipedsample from entering shuttle 324. For example, the walls of second end371 that define opening 372 may wipe excess sample from tip 204 as tip204 is inserted through opening 372. All or substantially all of thesample on the outer surface of tip 204 may be wiped away leaving onlysample disposed within tube 205. Tube 205 may hold, at most, apredetermined volume of sample, e.g., about 2 μl. Wiping excess samplefrom sample collection device 200 may enhance precision, accuracy,and/or consistency of analysis as, at most, a predetermined volume ofsample is inserted in sample preparation reservoir 317 in the mixingposition.

Referring now to FIGS. 10C through 10K, an exemplary process isdescribed for piercing the sealing material disposed over one or morereservoirs in the cartridge device via interaction between the samplecollection device and the seal piercer within the cartridge device. Asdescribed above, seal piercer 321 may include slider 322 and piercer323.

FIG. 10C is a top view of a portion of cartridge device 300 illustratinga possible orientation of piercing elements over the reservoirs. Piercer323 may have first piercing element 381 having a piercing end disposedover sample preparation reservoir 317, second piercing element 382having a piercing end disposed over wash reservoir 318, and/or thirdpiercing element 383 having a piercing end disposed over substratereservoir 319. In the pre-mixing state, piercing elements 381, 382, and383 are disposed over their respective reservoirs and have not piercedthe sealing material sealing fluid within the respective reservoirs.Piercing elements 381, 382, and 383 may be coupled (e.g., at an endopposite the piercing end) to the housing of the cartridge device.

FIG. 10D is a perspective view of sample collection device 200 withinthe input tunnel of cartridge device 300, with half the cartridgehousing removed to show slider 322 for clarity. Slider 322 may includefirst track 384 configured to engage and move first piercer 381 into apiercing position, second track 385 configured to engage and move secondpiercer 382 into a piercing position, and/or third track 386 configuredto engage and move third piercer 383 into a piercing position. As samplecollection device 200 is moved distally through input tunnel 301,preferably slider 322 does not move within the cartridge housing, andremains in a pre-vent position, until sample collection device 200securely engages slider 322. Slider 322 may securely engage samplecollection device 200 by temporarily or permanently coupling engager 380of slider 322 to engagement zone 209 of sample collection device 200.Engager 380 is disposed in input tunnel 301 (e.g., hanging below, atleast at times, slot 363 of FIG. 8A). Engager 380 may be sized to fitwithin a groove of engagement zone 209, e.g., protrusions and/orU-shaped, or to receive a protrusion of engagement zone 209.

FIG. 10E is a cross-sectional side view depicting the seal piercer inthe pre-piercing position and the shuttle in the pre-mixing position. Asshown, sample collection device 200 has been moved distally in inputtunnel 301 such that engager 380 of the slider has engaged engagementzone 209 of sample collection device 200 and distal sealing zone 208 ofsample collection device 200 has fluidicly sealed the opening of secondend 371 of shuttle 324. Preferably, first end 366 continues to fluidiclyseal fluid within sample preparation reservoir 317 at least until samplecollection device 200 fluidicly seals second end 371 of shuttle 324. Assuch, the one or more reagent balls housed by shuttle 324 and thecollected sample within shuttle 324 remain out of fluidic contact withthe fluid in sample preparation reservoir 317. Slider 322 and shuttle324 may be positioned within the cartridge housing such that engager 380of slider 322 engages engagement zone 209 of sample collection device200 at the same time, or approximately the same time, that distalsealing zone 208 of sample collection device contacts and fluidiclyseals second end 371 of shuttle 324. In such an embodiment, slider 322and/or shuttle 324 may not yet have been caused to move within cartridgedevice 300 at this time.

FIGS. 10F and 10G further illustrate positioning of the slider and thepiercer in the pre-venting position shown in FIG. 10E. As shown in FIG.10F, when engager 380 of slider 322 engages engagement zone 209 ofsample collection device 200 in the pre-venting position, piercingelements 381, 382, and 383 have not yet been deflected downwardly towardtheir respective reservoirs by slider 322. In such a pre-ventingposition, tracks 384, 385, and 386 may not yet contact piercing elements381, 382, and 383, respectively. As shown in FIG. 10G, in thepre-venting position, piercing element 381 is disposed above, but hasnot yet pierced, sealing material 320 which seals fluid within samplepreparation reservoir 317. In FIG. 10G, track 384 of slider 322 has notyet contacted piercing element 381 of piercer 323.

Referring now to FIG. 10H, sample collection device 200 is moved furtherdistally within input tunnel 301 from the pre-mixing and pre-ventingpositions towards the mixing and venting positions. As collector pushessample collection device 200 distally, shuttle 324 is partially movedwithin sample preparation reservoir 317. Shuttle 324 may be caused tomove within the cartridge by, for example, application of a forcegreater than a threshold force by sample collection device 200 (e.g., atdistal sealing zone 208) on shuttle 324 (e.g., at second end 371). Asshuttle 324 moves distally, first end 366 may unseal such that one ormore reagent balls within shuttle 324 and the collected sample withinshuttle 324 are exposed to fluid within sample preparation reservoir317. Advantageously, before first end 366 of shuttle 324 is unsealed,second end 371 of shuttle 324 is fluidicly sealed by sample collectiondevice 200 such that fluid from sample preparation reservoir 317 doesnot leak into input tunnel 301 beyond second end 371. In addition, asdistal movement of sample collection reservoir 200 causes the transitionfrom the pre-mixing position to the mixing position, second end 371 ofshuttle continuously fluidicly seals the fluid in sample preparationreservoir 317 from input tunnel 301.

As collector pushes sample collection device 200 distally, seal piercer321 also may be moved from the pre-venting position towards the ventingposition. Seal piercer 321 may be caused to move within the cartridgeby, for example, application of a force greater than a threshold forceby sample collection device 200 (e.g., at engagement zone 209) on sealpiercer 321 (e.g., at engager 380 of slider 322). As sample collectiondevice 200 is moved distally, seal piercer 321 causes the sealingmaterial over sample preparation reservoir 317, wash reservoir 318,and/or substrate reservoir 319 to be pierced to vent the fluid withinthe reservoir(s). Seal piercer 321 may be caused to move in a firstdirection, e.g., generally laterally in a direction generally parallelwith movement of sample collection device 200 within input tunnel 301,responsive to insertion of sample collection device 200 in input tunnel301 and in a second (different) direction, e.g., downwardly in agenerally vertical manner toward the respective reservoir, to pierceinto sealing material 320. For example, slider 322 may move in the firstdirection and piercer 323 may move in the second direction.

As shown in FIG. 10I, as sample collection device 200 causes slider 322to move distally in a direction generally parallel to movement of samplecollection device 200, slider 322 may contact piercer 323 to causepiercer 323 to move in a different direction, e.g., generallyperpendicular to movement of slider 322. As slider 322 moves distally,tracks 384, 385, and 386 of slider 322 may contact piercing elements381, 382, and 383, respectively, of piercer 323. Distal movement ofslider 322 may cause piercer 323 to pierce the sealing material to ventthe reservoir(s).

FIG. 10J illustrates piercing element 381 piercing sealing material 320over sample preparation reservoir 317 to vent sample preparationreservoir 317. As shown, track 384 contacts and moves piercing element381 downwardly into the piercing position.

FIG. 10K shows shuttle 324 in the mixing position and the seal piercerin the venting position. In the mixing position, first end 366 ofshuttle 324, the one or more reagent ball compartments of shuttlehousing one or more reagent balls, and/or the one or more samplecompartments housing the sample(s) to be analyzed may be disposed withinsample preparation reservoir 317. As explained above, shuttle 324 (e.g.,at second end 371) and sample collection device 200 (e.g., via tip 204inserted in opening 372 and distal sealing zone 208) also may fluidiclyseal sample preparation reservoir 317 in the mixing position such thatthe collected sample(s), the reagent ball(s), and the fluid withinsample preparation reservoir 317 are sealed within the reservoir. Inthis manner, the collected sample(s), the reagent ball(s), and the fluidmay be mixed within sample preparation reservoir 317.

Advantageously, a configuration where insertion of sample collectiondevice 200 causes sample preparation reservoir 317, wash reservoir 318,and/or substrate reservoir 319 to be vented ensures that thereservoir(s) remain fluidicly sealed prior to sample collection device200 insertion and facilitates drainage of the reservoir(s) into theanalysis channel when the outlet of the respective reservoir permitsfluid flow therethrough.

In the mixing position, seal piercer 321 may move out of the piercedholes to open the pierced holes and facilitate venting. As shown in FIG.10K, engagement zone 209 of sample collection device 200 may be moveddistally past engager 380 such that engager 380 disengages engagementzone 209 of sample collection device 200 in the venting position. Inaddition, in the mixing position, proximal sealing zone 207 of samplecollection device 200 is configured to seal input tunnel 301 at aperture302. In this manner, proximal sealing zone 207 provides additionalstructure to minimize or eliminate liquid leakage from cartridge device300, e.g., at aperture 302.

Cartridge device 300 may further include locking member 387 configuredto irreversibly lock sample collection device 200 within cartridgedevice 300. Locking member 387 may be biased inwardly in input tunnel301 such that a locking end of locking member 387 engages samplecollection device 200 in the mixing position. The locking end may lockto engagement zone 209. The locking end may be a protrusion sized to fitwithin a groove of engagement zone 209, as illustrated. Locking member387 also may define a portion of input channel 301, as illustrated, andmay be coupled to the cartridge housing at the end opposite its lockingend. Advantageously, locking sample collection device 200 (e.g.,longitudinally and/or axially) within input tunnel 301 promotes sealingof sample preparation reservoir 317 over time as sample collectiondevice 200 cannot be retracted once locked to facilitate safedisposability and consistency of testing because a user cannot pull outsample collection device 200 from cartridge device 300 inadvertentlyonce the test has started.

FIGS. 10L and 10M further depict sample collection device 200 in themixing position within cartridge device 300 (FIG. 10L) and internalcomponent 316 (FIG. 10M) for clarity.

Referring now to FIGS. 10N, 10O, and 10P, a process for enhanced mixingof the contents within sample preparation reservoir 317 is described. Inthe mixing position, the fluid within sample preparation reservoir 317is mixed with the collected sample and one or more reagent balls (ifprovided). Mixing may be enhanced via sonicator element 327 which may bea piezoelectric transducer such as a piezoceramic disc. Sonicatorelement 327 is configured to vibrate responsive to electrical signals(e.g., transmitted from the reader device) to further mix the contentswithin sample preparation reservoir 317. For example, sonicator element327 may facilitate mixing of fluid held in sample preparation reservoir317, which may be pre-filled and/or filled during the mixing process,with reagent ball 375 and the collected sample. Sonicator element 327may cause the fluid to flow in a wave pattern as shown in FIGS. 10O and10P. Such a wave pattern may be between compartments defined by theshuttle, e.g., between reagent ball compartment 367 and samplecompartment 370. For example, sonicator element 327 may cause the fluidto flow in one or more directions (e.g., generally up and down as shownin FIG. 10O) within reagent ball compartment 367 during portions of thewave cycle. Such flow is expected to speed up, and enhance, mixingwithin sample preparation reservoir 317. The wave motion of fluid withinsample preparation reservoir 317 also may facilitate removal of samplefrom the distal portion of sample collection device 200 to enhancemixing and homogenization.

Sonicator element 327 may be configured to emit acoustic waves to movethe fluid in sample preparation reservoir 317 in the wave patternbetween reagent ball compartment 367 and sample compartment 370 to mixthe fluid in sample preparation reservoir 317. Such mixing may create afluid mix of the sample, fluid from the reservoir, and the dissolvedreagent ball(s). The acoustic emissions from sonicator element 327 mayboth heat fluid within sample preparation reservoir 317 and mix thecontents of sample preparation reservoir 317 at the macro and the microlevel for amplification such as isothermal amplification. The reagentball(s) may include, for example, polymerases, primers, and signalingagents for isothermal amplification. Shuttle 324 may have compartmentdivider 368, which may be a flange, configured to divide reagent ballcompartment 367 and sample compartment 370. The fluid flowing aroundcompartment divider 368 may facilitate formation of the wave pattern.Compartment divider 368 may have a slot configured to permit the fluidto flow through compartment divider 368 via the slot during mixing.Sonicator element 327 may form a wall, e.g., part of the bottom wall, ofsample preparation reservoir 317 and may be positioned off-center ofsample preparation reservoir 317 to facilitate mixing of the fluidwithin sample preparation reservoir 317. For example, sonicator element327 may be positioned off-center relative to a center axis of samplepreparation reservoir 317 running perpendicular to a longitudinal axisrunning through the center of the input tunnel of the cartridge. Suchoff-center positioning of sonicator element 327 also facilitatesenhanced mixing. Sonicator element 327 may be electrically coupled to aprinted circuit board via one or more spring contacts as described indetail below.

Referring now to FIGS. 11A through 11E, an alternative seal piercer isshown. FIGS. 11A and 11B show seal piercer 321′ in a pre-venting andpre-contact position. Seal piercer 321′ includes slider 322′ slidablycoupled to piercer 323′ throughout the piercing process. Seal piercer321′ may further include post 388 configured to press contact switch 389of circuit board 331′. Depression of contact switch 389 may complete acircuit such that electrical signals may be transmitted, e.g., to thereader device and/or the computing device running the software-baseduser interface system. In this manner, proper insertion of samplecollection device 200′ within the input tunnel generates an electricalsignal that may be transmitted to the reader device and/or computingdevice to notify the reader device and/or computing device of suchproper insertion. Post 388 may be coupled to piercer 323′ or may beintegral with piercer 323′. As sample collection device 200′ causesslider 322′ to move distally in a direction generally parallel tomovement of sample collection device 200′ (e.g., by force applied byengagement zone 209′ and/or engagement zone 210 to engager 380′), slider322′ may cause piercer 323/post 388 to move in a different direction,e.g., generally perpendicular to movement of slider 322′. Slider 322′may have angled face 390 configured to contact angled face 391 ofpiercer 323′ as distal movement of sample collection device 200′ causesdistal movement of slider 322′. Continuing distal movement of samplecollection device 200′, and thereby distal movement of slider 322′,causes piercer 323′ and post 388 to be moved downwardly such thatpiercer 323′ pierces the sealing material on the respective reservoir(s)and post 388 activates contact switch 389 as shown in FIGS. 11D and 11Ein the contact position.

After contact with contact switch 389 and piercing of the sealingmaterial, seal piercer 321′ may move such that post 388 no longerdepresses contact switch 389 and piercer 323′ moves out of the hole(s)pierced in the sealing material to vent the respective reservoir(s).Insertion of the sample collection device within the input tunnel maycause the seal piercer to pierce the sealing material before a shuttlebegins movement from the pre-mixing position to the mixing position orduring movement from the pre-mixing position to the mixing position.

FIGS. 11A to 11E illustrate sonicator element 327′ coupled to springcontact 392. Spring contact 392 is coupled to circuit board 331′ and isa conductor such that sonicator element 327′ is electrically coupled tocircuit board 331′ via spring contact 392. Beneficially, spring contact392 absorbs movement of sonicator element 327′ such that circuit board331′ vibrates minimally in a suitable manner when sonicator element 327′is activated, e.g., responsive to signals transmitted by the readerdevice, and spring contact 392 permits ease of assembly andreproducibility compared to soldering which may adversely impactsonicator element 327′.

As will be readily understood to one skilled in the art, while FIGS. 11Aand 11D do not depict a shuttle and/or reagent balls within the inputtunnel, such features may be included in these embodiments.

Referring now to FIGS. 12A through 12E, an alternative configuration forcollecting and analyzing a fluid sample is described. Cartridge device300″ may be constructed similarly to cartridge device 300 describedabove except cartridge device 300″ may be modified for enhancedcollection of relatively large amounts of fluid. In addition, samplecollection device 200″ may be constructed similarly to sample collectiondevice 200 described above except sample collection device 200″ may havea modified collection area for enhanced collection of relatively largeamounts of fluid. For example, sample collection device 200″ andcartridge device 300″ may be particularly useful for collecting andanalyzing saliva, blood, plasma, urine, or the like.

Sample collection device 200″ may include modified distal portion 201″for enhanced collection of relatively large amounts of fluid (e.g.,about 10-100 microliters) that may include distal sealing zone 208″,wicking portion 211, intermediate sealing zone 212, and shroud 213.Distal portion 201″ of sample collection device 200″ is adapted to beexposed to a sample, preferably a liquid sample, to absorb at least aportion of the sample, and to be compressed to expel the collectedsample from distal portion 201″ into cartridge device 300″ for analysisof the expelled sample. Wicking portion 211 is configured to wick andabsorb a sample and may be formed from a wicking material. Wickingportion 211 may be coupled to intermediate sealing zone 212.Intermediate sealing zone 212 may be slidably disposed within shroud 213and may include one or more sealing members, e.g., O-rings, configuredto create a liquid tight seal that reduces or prevents fluid absorbed onwicking portion 211 from moving proximally within shroud 213 beyondintermediate sealing zone 212. Wicking portion 211 is at least partiallydisposed within shroud 213 and may slide within a lumen of shroud 213.Wicking portion 211 may be configured to become transparent when exposedto fluid such that increasing amounts of wicking portion 211 becometransparent as increasing amounts of sample fluid are collected. Samplecollection device 200″ may include sample collection indicator 214configured to visually alert a collector based on a volume of samplefluid that has been collected. In one embodiment, sample collectionindicator 214 visually alerts a collector as the volume of samplecollected increases and, optionally, that at least a predeterminedvolume of sample fluid has been collected. For example, samplecollection indicator 214 may change color when the predetermined volumeof sample, or more, has been collected. As another example, anincreasing amount of sample collection indicator 214 may become visibleas the volume of sample collected increases. For example, samplecollection indicator 214 may be a colored thread embedded in wickingportion 211 that becomes increasingly visually exposed as increasingvolumes of collected fluid sample cause the surrounding wicking materialto turn more transparent such that a collector may monitor progress offluid collection and determine when a sufficient volume of sample hasbeen collected. In one embodiment, sample collection indicator 214includes a transparent area on shroud 213. In addition, oralternatively, sample collection indicator 214 may change color as thevolume of sample collected increases.

Shuttle 324″ may include first end 366″, reagent ball compartment 367″,compartment divider 368″, and sample compartment 370″ constructedsimilarly to those respective components described above. Preferably,compartment divider 368″ does not have a slot, similar to compartmentdivider 368′, such that compartment divider 368″ fluidicly seals reagentball compartment 367″ from sample compartment 370″ in the pre-mixingposition. Second end 371″ of shuttle 324″ may be modified to includedistal flange 393 and proximal flange 394 having cavity 395. Inaddition, unlike opening 372 of shuttle 324, opening 372″ is configuredto permit flow of expelled sample compressed from sample collectiondevice 200″ to sample compartment 370″, but not a portion of samplecollection device 200″. Second end 371″ (e.g., at distal flange 393) maybe configured to fluidicly seal sample compartment 370″ in both thepre-mixing position and the mixing position and continuously during thetransition therebetween. Distal flange 393 may include one or moresealing members, e.g., O-rings, configured to create a liquid tight sealthat reduces or prevents fluid from flowing proximally out of samplecompartment 370″. In addition, compartment divider 368″ may beconfigured to fluidicly seal sample compartment 370″ in the pre-mixingposition. Compartment divider 368″ may include one or more sealingmembers, e.g., O-rings, configured to create a liquid tight seal thatreduces or prevents fluid from flowing distally out of samplecompartment 370″.

Prior to insertion within input tunnel 301″ of cartridge device 300″,sample collection device 200″ is exposed to a sample, e.g., within aninner cheek, the throat, the mouth, a nasal passageway, an ear, fromurine, from blood, from plasma, from saliva, etc. Wicking portion 211 ofsample collection device 200″ is designed to retain some of the sampleto permit analysis of the presence, absence, and/or quantity of one ormore target analytes within the sample using cartridge device 300″ andthe reader device. Cartridge device 300″ may be electrically coupled tothe reader device before or after sample collection device 200″ isinserted in cartridge device 300″.

Referring to FIG. 12A, the distal end of sample collection device 200″is first inserted into aperture 302″ defining the opening of inputtunnel 301″. Shuttle 324″ is disposed within input tunnel 301″ in apre-mixing position wherein first end 366″ of shuttle 324″ forms a wallof sample preparation reservoir 317″ to fluidicly seal fluid withinsample preparation reservoir 317″. In this pre-mixing position, reagentball 375″ housed by shuttle 324″ is within input tunnel 301″ and notexposed to fluid within sample preparation reservoir 317″.

Referring to FIG. 12B, as sample collection device 200″ moves distallyin input tunnel 301″, sample collection device 200″ may contact shuttle324″. For example, the distal end and/or distal sealing zone 208″ maycontact second end 371″ (e.g., at cavity 395 and/or proximal flange 394)as illustrated. Cavity 395 may be sized slightly larger than the outersurface of wicking portion 211 such that the distal end of wickingportion 211 fits snuggly within cavity 395. Distal sealing zone 208″ maybe configured to fluidicly seal sample collection device 200″ to shuttle324″ such that fluid expelled from sample collection device 200″ travelsinto sample compartment 370″. In this pre-mixing position, samplecompartment 370″ is within input tunnel 301″ and not exposed to fluidwithin sample preparation reservoir 317″.

As shown in FIG. 12C, as sample collection device 200″ moves furtherdistally in input tunnel 301″, sample collection device 200″ may expelcollected fluid sample into sample compartment 370″ of shuttle 324″,e.g., through opening 372″ of second end 371″. As sample collectiondevice 200″ is moved distally, wicking portion 211 may be compressed toexpel collected sample and the distal end of wicking portion 211 mayremain substantially in position during compression. Intermediatesealing zone 212 and/or shroud 213 may move distally in proportion tomovement of the handle of sample collection device 200″ during suchcompression. Preferably, as wicking portion 211 is compressed and sampleis expelled therefrom, the expelled sample travels through opening 372″into sample compartment 370″. During compression, shuttle 324″ mayremain substantially in position within input tunnel 301″ and first end366″ of shuttle 324″ may continue to form a wall of sample preparationreservoir 317″ to seal fluid within sample preparation reservoir 317″.Cartridge device 300″ may include proximal ledge 396 configured to holdshuttle 324″ in the pre-mixing position during compression of wickingportion 211. Proximal ledge 396 may engage proximal flange 394 to holdshuttle 324″ in position.

A predetermined volume of sample, at most, may be configured to be heldin sample compartment 370″. Cartridge device 300″ may include overflowcompartment 397 and overflow lumen 398. Overflow compartment 397 andoverflow lumen 398 may be part of the cartridge housing or the internalcomponent within the cartridge. If the amount of sample introduced intosample compartment 370″ exceeds the predetermined volume, e.g., exceedsabout 20 μl, the excess sample may travel to overflow compartment 397fluidicly connected to sample compartment 370″, e.g., via overflow lumen398. Limiting the volume of sample within sample compartment 370″ mayenhance precision, accuracy, and/or consistency of analysis as, at most,a predetermined volume of sample is inserted in sample preparationreservoir 317 in the mixing position. Overflow compartment 397 may beotherwise sealed to prevent or reduce leakage of excess sample withinoverflow compartment 397.

FIG. 12D shows sample collection device 200″ and cartridge device 300″in the mixing position and FIG. 12E shows a close up view of a portionof FIG. 12D for clarity. As collector pushes sample collection device200″ distally from the pre-mixing position to the mixing position,shuttle 324″ is partially moved within sample preparation reservoir317″. Application of a force greater than a threshold force on shuttle324″ (e.g., at second end 371″ and preferably within cavity 395) bysample collection device 200″ (at distal sealing portion 208″ and/or thedistal end of wicking material 211) may cause shuttle 324″ to move fromthe pre-mixing position to the mixing position wherein the expelledsample in sample compartment 370″ and reagent ball 375″ are mixed withinfluid of sample preparation reservoir 317″. For example, the thresholdforce may be the force required to push shuttle 324″ distally pastproximal ledge 396. As shuttle 324″ moves distally, first end 366″ mayunseal such that one or more reagent balls within shuttle 324″ and thecollected sample within shuttle 324″ are exposed to fluid within samplepreparation reservoir 317″. Advantageously, before first end 366″ ofshuttle 324″ is unsealed, second end 371″ of shuttle 324″ is fluidiclysealed by sample collection device 200″ such that fluid from samplepreparation reservoir 317″ does not leak into input tunnel 301″ beyondsecond end 371″ and/or intermediate sealing zone 212. In addition, asdistal movement of sample collection device 200″ causes the transitionfrom the pre-mixing position to the mixing position, distal flange 393of shuttle 324″ continuously fluidicly seals the fluid in samplepreparation reservoir 317″ from input tunnel 301″. Cartridge device 300″may include distal ledge 399 configured to hold shuttle 324″ in themixing position and to prevent further distal movement. Distal ledge 399may engage proximal flange 394 to hold shuttle 324″ in position.

In the mixing position, first end 366″ of shuttle 324″, the one or morereagent ball compartments of shuttle housing one or more reagent balls,and/or the one or more sample compartments housing the sample(s) to beanalyzed may be disposed within sample preparation reservoir 317″. Asexplained above, shuttle 324″ (e.g., at second end 371″) and samplecollection device 200″ (e.g., via distal sealing zone 208″ inserted incavity 395) also may fluidicly seal sample preparation reservoir 317″ inthe mixing position such that the collected sample(s), the reagentball(s), and the fluid within sample preparation reservoir 317″ aresealed within the reservoir. In this manner, the collected sample(s),the reagent ball(s), and the fluid may be mixed within samplepreparation reservoir 317″.

Cartridge device 300″ may further include locking member 387″ configuredto irreversibly lock sample collection device 200″ within cartridgedevice 300″. Locking member 387″ may be biased inwardly in input tunnel301″ such that a locking end of locking member 387″ engages samplecollection device 200″ in the mixing position. The locking end may lockto an engagement zone of sample collection device 200″. The locking endmay be a protrusion sized to fit within a groove on the shaft of samplecollection device 200″ or within a groove on shroud 213, as illustrated.Locking member 387″ also may define a portion of input channel 301″, asillustrated, and may be coupled to the cartridge housing at the endopposite its locking end. Advantageously, locking sample collectiondevice 200″ (e.g., longitudinally and/or axially) within input tunnel301″ promotes sealing of sample preparation reservoir 317″ over time assample collection device 200″ cannot be retracted once locked tofacilitate safe disposability and consistency of testing because a usercannot pull out sample collection device 200″ from cartridge device 300″inadvertently once the test has started.

As will be readily understood to one skilled in the art, while FIGS. 12Athrough 12E do not depict a seal piercer in the cartridge device, a sealpiercer may be included in these embodiments. For example, samplecollection device 200″ (e.g., at distal sealing zone 208″) may contactthe seal piercer to move the seal piercer from the pre-venting positionto the venting position in a manner described above with respect toFIGS. 10A to 10J and/or FIGS. 11A to 11E. Sample collection device 200″may contact and move the seal piercer from the pre-venting to theventing position before, during, and/or after contacting shuttle 324″and moving shuttle 324″ from the pre-mixing to the mixing position. Inaddition, insertion of sample collection device 200″ may causeactivation of a contact switch as described above with respect to FIGS.11A to 11E. For example, sample collection device 200″ may causemovement of a seal piercer which in turn causes activation of thecontact switch.

Referring now to FIG. 13A, an alternative cartridge for analyzing asample is described. Cartridge device 300′″ may be constructed similarlyto cartridge device 300 and/or cartridge device 300″ described abovewherein like components are identified by like-primed reference numbers.Cartridge device 300′″ is a universal configuration that includescomponents that may be used for different types of samples. For example,many components within cartridge device 300′″ may be used for varioustypes of samples without modification and, in some embodiments, only theshuttle, collet, and reagent ball(s) may be varied for analysis ofdifferent indications. In this manner, cartridge device 300′″ may beuniversal and the shuttle, collet, and/or reagent ball(s) may beselected for use in cartridge device 300′″ based on the targetanalyte(s) to be analyzed. For example, cartridge device 300′″ may befitted with a shuttle designed for relatively small sample collection,e.g., a nasal passageway, an ear, blood, such as shuttle 324 or 324′described above with respect to FIGS. 9A and 9B, and a collet alsodesigned for relatively small sample collection or cartridge device300′″ may be fitted with a shuttle designed for relatively large fluidsample collection, e.g., saliva, blood, plasma, urine, such as shuttle324″ described above with respect to FIGS. 12A through 12E and a colletalso designed for relatively large fluid sample collection. Cartridgedevice 300′″ may be further fitted with a reagent ball(s) intended toidentify different target analytes that may be indicative of, forexample, inflammation, influenza, testosterone, fertility, HIV, orVitamin D. Accordingly, cartridge device 300′″ is highly interchangeablyfor different types of samples and indications which reduces costs andmanufacturing burdens.

In FIG. 13A, an exploded view of cartridge device 300′″ is shown.Cartridge device 300′″ may include internal component 316′″—which mayinclude sample preparation reservoir 317′″, wash reservoir 318′″,substrate reservoir 319′″, input tunnel component 326′″, overflowcompartment 397′″, and/or posts 610 and 612—sealing material 320′″, sealpiercer 321′″, shuttle 324′″ having first end 366′″ and sealing members614, desiccant 325′″, sonicator element 327′″, absorbent pad 328′″,layer 329′″, analysis channel 330′″, circuit board 331′″, memory 332′″,sensor 338′″, heating elements, contact switch 389′″, spring contacts392′″, reagent ball 375′″, temperature sensor 616, and/or collet 618.The internal components may be disposed within housing 304′″, e.g.,between first and second cover components 310′″ and 311′″.Alternatively, one or more internal components may be disposed withinone housing while other internal components may be disposed withinanother housing(s). In the case of multiple housings, such separatehousing may be configured to couple to one another.

In FIG. 13A, shuttle 324′″ is illustrated as being substantially similarto shuttle 324″ of FIGS. 12A through 12E although a shuttle like that ofshuttle 324 in FIG. 9A or shuttle 324′ in FIG. 9B may be used dependingon the type of sample to be collected. Collet 618 also may besubstituted for collet 618′ described below based on the type of sampleto be collected. Various reagent ball(s) 375′″ may be substituted incartridge device 300′″ as well.

Posts 610 and 612 are configured to be coupled to seal piercer 321′″ andto permit seal piercer 321′″ to be moved from the pre-venting positionto the venting position. Posts 610 and 612 may be integrally formed withinternal component 316′″ or may be separate pieces.

Temperature sensor 616 may be configured to sense temperature indicativeof temperature of fluid in a reservoir, e.g., sample preparationreservoir 317′″. For example, temperature sensor 616 may sensetemperature changes adjacent the reservoir that are indicative oftemperature in the reservoir. Temperature sensor 616 may be a thermistorand may be disposed on circuit board 331′″ to permit electrical couplingwith the reader device via one or more leads in circuit board 331′″.Preferably, temperature sensor 616 is positioned adjacent sonicatorelement 327′″ on circuit board 331′″ such that temperature sensor 616senses temperature during mixing within sample preparation reservoir317′″ via sonicator element 327′″. Advantageously, temperatureindicative of temperature in sample preparation reservoir 317′″ may bemonitored during mixing of the fluid in the reservoir with reagents fromreagent ball(s) and the sample, to ensure that temperature within samplepreparation reservoir 317′″ is within a predetermined range. If outsidethe predetermined range, emission of acoustic waves into samplepreparation reservoir 317′″ via sonicator element 327′″ may be modified,e.g., responsive to electrical signals sent by the reader to thesonicator element 327′″. Temperature sensor 616 may generate a signalindicative of temperature of the fluid in the reservoir which may betransmitted to the reader for processing via leads in circuit board331′″. Temperature sensor 616 may be positioned beneath sonicatorelement 327′″ on circuit board 331′″ and adjacent to one or more springcontacts, e.g., between first and second spring contacts 392′″.

Collet 618 is preferably disposed in input tunnel 301′″ between aperture302′″ and sample preparation reservoir 317′″. Collet 618 also may bedisposed at least partially proximal to shuttle 324′″ in input tunnel301′″. For example, in the pre-mixing position, an end, e.g., the secondend, of the shuttle may be disposed within collet 618. Collet 618 alsomay be configured to hold shuttle 324′″ in the pre-mixing position andto decouple from shuttle 324′″ during insertion of the sample collectiondevice in input tunnel 301′″. In this manner, collet 618 may holdshuttle 324′″ in the pre-mixing state until force applied from thesample collection device decouples collet 618 from shuttle 324′″ suchthat shuttle 324′″ moves from the pre-mixing position to the mixingposition while collet 618 remains in place within input tunnel 301′″.Collet 618 has a lumen therethrough sized to permit insertion of thedistal portion of the sample collection device. Collet 618 may have agenerally tubular shape as illustrated in FIG. 13A. Collet 618 also maybe configured to activate contact switch 389′″. For example, collet 618may activate contact switch 389′″ responsive to a force applied oncollet 618 by the sample collection device during insertion of thesample collection device in input tunnel 301′″.

Referring now to FIGS. 13B through 13SS, various exemplary samplecollection devices are illustrated that may be used in detection system100.

Referring to FIG. 13B, sample collection device 200′″ may be constructedsimilarly to sample collection device 200″ described above except samplecollection device 200′″ may have a modified shaft for locking samplecollection device 200′″ within a cartridge during partial and fullinsertion of sample collection device 200′″. For example, samplecollection device 200′″ illustratively includes engagement zone 209′″having a plurality of grooves and protrusions distal to, and spacedapart from, proximal sealing zone 207′″. The plurality of grooves andprotrusions are configured for engagement with one or more components ofthe cartridge device for irretractability of sample collection device200′″ during partial and full insertion of sample collection device200′″ within the cartridge. For example, the cartridge may sequentiallyengage grooves of the multiplicity of grooves in engagement zone 209′″in a distal to proximal direction as sample collection device 200′″ ismoved distally in the input tunnel. Engagement zone 209′″ may beconfigured to be coupled, permanently or temporarily, to the sealpiercer of the cartridge device to move the seal piercer within thecartridge device responsive to movement of sample collection device200′″. In addition or alternatively, engagement zone 209′″ may beconfigured to activate a contact switch during sample collection deviceinsertion. For example, shoulder 220 at the distal end of engagementzone 209′″ may be configured to be coupled to the seal piercer and/or toactivate the contact switch.

Like sample collection device 200″, sample collection device 200′″ mayinclude modified distal portion 201′″ for enhanced collection ofrelatively large amounts of fluid (e.g., about 10-100 microliters,preferably about 20 microliters) that may include distal sealing zone208′″, wicking portion 211′″, intermediate sealing zone, and/or shroud213′″. Sample collection device 200′″ also may include proximal portion202′″, shaft 203′″ extending between distal portion 201′″ and proximalportion 202′″, handle 206′″, proximal sealing zone 207′″, and/orengagement zone 209′″ having shoulder 220 similar to the like primedreferences described above. Sample collection device 200′″ is configuredfor full or partial insertion within cartridge device 300′″ after samplecollection. Sample collection device 200′″ and cartridge device 300′″may be particularly useful for collecting and analyzing saliva, blood,plasma, urine, or the like. Shoulder 220 of engagement zone 209′″preferably extends further from the longitudinal axis of samplecollection device 200′″ than a shoulder of distal sealing zone 208′″such that shoulder 220 contacts and moves the seal piercer to vent oneor more reservoirs within cartridge device 300′″ and/or contacts thecollet to activate the contact switch while the shoulder of distalsealing zone 208′″ may be sized to move distally through the inputtunnel without moving the seal piercer and/or without activating thecontact switch.

FIGS. 13C, 13D, 13E, 13F, 13G, and 13H are back, side, front, back,side, and front views, respectively, of sample collection device 200′″.

Referring to FIG. 13I, sample collection device 200″″ may be constructedsimilarly to sample collection device 200 described above except samplecollection device 200″″ may have engagement zone 209″″ similar toengagement zone 209′″ of FIG. 13B and tip 204″″ does not include a tube.Tip 204″″′ may have a rounded end as illustrated and may be configuredto collect a sample from any desired region or location, although tip204″″ may be particularly useful when collecting a sample from a nasalarea. FIGS. 13J, 13K, 13L, 13M, 13N, and 13O are back, side, front,back, side, and front views, respectively, of sample collection device200″″.

Referring to FIG. 13P, sample collection device 200′″″ may beconstructed similarly to sample collection device 200″″ shown in FIG.131 except tip 204′″″ of sample collection device 200′″″ includes tube205′″″ (like the tube shown in FIGS. 2A and 2B). Distal portion 201′″″,including tip 204′″″, is configured to be exposed to a sample such that,at most, a predetermined volume of the sample (e.g., less than 10microliters, preferably about 2 microliters) is disposed in tube 205′″″for analysis. Collection of a predetermined volume of the sample isexpected to promote accuracy of analyte analysis as a substantiallyknown quantity of the sample will be analyzed. Tip 204′″″ may have arounded end as illustrated and may be configured to collect a samplefrom any desired region or location, although tip 204′″″ may beparticularly useful when collecting a sample of blood. FIGS. 13Q, 13R,13S, 13T, 13U, 13V, and 13W are side, back, side, front, back, side, andfront views, respectively, of sample collection device 200′″″.

Referring to FIG. 13X, sample collection device 200″″″ may beconstructed similarly to sample collection device 200′″″ shown in FIG.13P except tip 204″″″ of sample collection device 200″″″ includes slot222 rather than a tube. Distal portion 201″″″, including tip 204″″″, isconfigured to be exposed to a sample such that, at most, a predeterminedvolume of the sample (e.g., less than 10 microliters, preferably about 5microliters) is disposed in slot 222 for analysis. Collection of apredetermined volume of the sample is expected to promote accuracy ofanalyte analysis as a substantially known quantity of the sample will beanalyzed. Tip 204″″″ may have a rounded end as illustrated and may beconfigured to collect a sample from any desired region or location,although tip 204″″″ may be particularly useful when collecting a sampleof blood. FIGS. 13Y, 13Z, 13AA, 13BB, 13CC, 13DD, and 13EE are side,back, side, front, back, side, and front views, respectively, of samplecollection device 200″″″.

Referring to FIG. 13FF, sample collection device 200′″″″ may beconstructed similarly to sample collection device 200′″″ shown in FIG.13P except tip 204′″″″ of sample collection device 200′″″″ includes ring224 rather than a tube. Distal portion 201′″″″, including tip 204′″″″,is configured to be exposed to a sample such that, at most, apredetermined volume of the sample (e.g., less than 10 microliters,preferably about 2 microliters) is disposed in a groove formed by ring224 for analysis. Collection of a predetermined volume of the sample isexpected to promote accuracy of analyte analysis as a substantiallyknown quantity of the sample will be analyzed. Tip 204′″″″ may have arounded end as illustrated and may be configured to collect a samplefrom any desired region or location, although tip 204′″″″ may beparticularly useful when collecting a sample of blood. FIGS. 13GG, 13HH,13II, 13JJ, 13KK, and 13LL are back, side, front, back, side, and frontviews, respectively, of sample collection device 200′″″″.

Referring to FIG. 13MM, sample collection device 200″″″″ may beconstructed similarly to sample collection device 200′″″ shown in FIG.13P except tip 204″″″″ of sample collection device 200″″″″ includesfirst ring 226 and second ring 228 rather than a tube. Distal portion201″″″″, including tip 204″″″″, is configured to be exposed to a samplesuch that, at most, a predetermined volume of the sample (e.g., lessthan 10 microliters, preferably about 5 microliters) is disposed in agroove formed by first ring 226 and a groove formed by second ring 228for analysis. Collection of a predetermined volume of the sample isexpected to promote accuracy of analyte analysis as a substantiallyknown quantity of the sample will be analyzed. Tip 204″″″″ may have arounded end as illustrated and may be configured to collect a samplefrom any desired region or location, although tip 204″″″″ may beparticularly useful when collecting a sample of blood. FIGS. 13NN, 13OO,13PP, 13QQ, 13RR, and 13SS are back, side, front, back, side, and frontviews, respectively, of sample collection device 200″″″″.

Referring now to FIGS. 14A and 14B, an exemplary collet for use in acartridge is described. Collet 618 may be specifically designed forrelatively large fluid sample collection, e.g., saliva, blood, plasma,urine, such as when the sample is compressed from the distal portion ofthe sample collection device as described above with respect to FIGS.12A through 12E, FIG. 13B, and below. Collet 618 may include proximalend 620, distal end 622, and lumen 624 extending between ends 620 and622. Lumen 624 may be sized to permit insertion of the distal portion ofa sample collection device into lumen 624. Collet 618 is positioned inthe input tunnel such that the distal portion of the sample collectiondevice first enters lumen 624 at proximal end 620. Collet 618 also mayinclude slot 626, e.g., at upper surface of collet 618. Slot 626 issized to receive a portion of the seal piercer therethrough. Forexample, the engager of the seal piercer may extend through the slotinto the input tunnel to permit contact between the seal piercer and thesample collection device.

Lumen 624 of collet 618 also may be sized to receive a portion of theshuttle therein. For example, the proximal end of the shuttle may bedisposed in lumen 624 through distal end 622 of collet 618. Collet 618also may be configured to hold the shuttle in the input tunnel in thepre-mixing position. For example, collet 618 may include one or morelocking arms configured to couple collet 618 to the shuttle in thepre-mixing position. Illustratively, collet 618 includes first lockingarm 628 and second locking arm 630 at opposing lateral sides of collet618. Advantageously, when using a sample collection device having acompressible distal portion for collecting a fluid sample, collet 618may hold the shuttle in place in the input tunnel during compression ofthe distal portion to expel sample fluid into the shuttle. Each lockingarm may include a ramp and a protrusion, shown as ramp 632 andprotrusion 634 for locking arm 630 in FIG. 14B. Protrusion 634 may becoupled to the shuttle to hold the shuttle in place in the pre-mixingposition. For example, the protrusions may hold the proximal flange ofthe shuttle during compression of the distal portion of the samplecollection device. First and second locking arms 628 and 630 also maydecouple from the shuttle during insertion of the sample collectiondevice in the input tunnel 301. First and second locking arms 628 and630 may be deflected to decouple first and second locking arms 628 and630 from the shuttle responsive to a force applied on first and secondlocking arms 628 and 630 by the sample collection device duringinsertion of the sample collection device in the input tunnel. Forexample, the shoulder of the sample collection device may contact theramp(s) of the locking arm(s) and cause the protrusion(s) to deflectoutwardly as the sample collection device moves distally along theramp(s) and in the input tunnel. The ramp(s) are shaped such that theprotrusion(s) decouple from the proximal flange of the shuttle to unlockthe shuttle and permit the shuttle to move from the pre-mixing positionto the mixing position where the shuttle is partially disposed withinthe sample preparation reservoir.

Collet 618 may include deflector portion 636 configured to deflect toactivate a contact switch within the cartridge. Preferably, deflectorportion 636 is disposed on a bottom face of collet 618 and positionedabove the contact switch in the input tunnel. Deflector portion 636 maydeflect to activate the contact switch responsive to a force applied ondeflector portion 636 by the sample collection device during insertionof the sample collection device in the input tunnel. For example, theshoulder of the sample collection device may contact deflector portion636 and force deflector portion 636 downwardly as the sample collectiondevice moves distally in the input tunnel to activate the contactswitch. Illustratively, deflector portion 636 is an arm configured todeflect downwardly.

Referring now to FIGS. 14C and 14D, an alternative collet for use in acartridge is described. Collet 618′ may be specifically designed forrelatively small sample collection, e.g., a nasal passageway, an ear,blood, such as when the sample need not be compressed from the distalportion of the sample collection device. As will be appreciated whencomparing FIGS. 14C and 14D to FIGS. 14A and 14B, collet 618′ is similarto collet 618 expect collet 618′ does not have locking arms. Collet 618′has one or more protrusions 637 disposed at distal end 622′ andconfigured to contact the proximal end of the shuttle in the pre-mixingposition. For example, the one or more protrusions 637 may contact asealing member, e.g., O-ring, at second end 371, 371′ of shuttle 324,324′ to retain the sealing member in position. The one or moreprotrusions may have lead-in angles configured to guide the distalportion of the sample collection device into the opening at the secondend of the shuttle.

Referring now to FIGS. 15A through 15D, cartridge 300′″ is shown invarious positions with the upper surface of the housing removed forclarity. FIGS. 15A and 15B are cross-sectional views through the centerof the input tunnel for additional clarity. In FIG. 15A, cartridge 300′″is shown in the pre-mixing, pre-venting, storage position where a samplecollection device has not yet been inserted in input tunnel 301′″. Asshown, seal piercer 321′″ is in the pre-venting position and has not yetpierced seal material 320′″ over the reservoirs, the proximal end ofshuttle 324′″ is disposed within the distal end of collet 618 while thedistal end of shuttle 324′″ forms a wall of sample preparation reservoir317′″, and deflector portion 636 of collet 618 is in the pre-deflectionposition where deflector portion 636 does not activate contact switch389′″. In FIG. 15B, cartridge 300′″ is shown in the mixing, venting,analysis position where the sample collection device is fully insertedin input tunnel 301′″. As shown, seal piercer 321′″ is in the ventingposition and has pierced seal material 320′″ over each of thereservoirs, shuttle 324′″ has moved distally from collet 618 such thatthe sample and reagent ball 375′″ are mixing with the fluid in samplepreparation reservoir 317′″, deflector portion 636 of collet 618 is inthe deflection position where deflector portion 636 has activatedcontact switch 389′″, and locking members 387′″ have locked the samplecollection device in input tunnel 301′″. In FIG. 15C, cartridge 300′″ isstill in the pre-mixing, pre-venting position as the sample collectiondevice has only been partially inserted in input tunnel 301′″. FIG. 15Dshows cartridge 300′″ in the venting position.

Referring back to FIG. 15A, an exemplary process is described forpiercing the sealing material disposed over one or more reservoirs inthe cartridge device via interaction between the sample collectiondevice and the seal piercer within the cartridge device.

Seal piercer 321′″ is configured to pierce sealing material 320′″ tovent the fluid in sample preparation reservoir 317′″, wash reservoir318′″, and/or substrate reservoir 319′″. Preferably, seal piercer 321′″is configured to pierce sealing material 320′″ over the reservoirssequentially to reduce resistance on the sample collection device duringpiercing. Seal piercer 321′″ may be configured to be contacted by thedistal portion, e.g., at a shoulder, of a sample collection devicewithin input tunnel 301′″ and to move within housing 304′″, responsiveto force applied by the sample collection device, to cause sealingmaterial 320′″ to be pierced to vent the fluid in sample preparationreservoir 317′″, wash reservoir 318′″, and/or substrate reservoir 319′″.Illustratively, seal piercer 321′″ is a single piece. Seal piercer 321′″is disposed within housing 304′″ and may be partially disposed withininput tunnel 301′″. For example, engager 380′″ of seal piercer 321′″ maybe disposed within input tunnel 301′″, e.g., through slot 626 of collet618. Seal piercer 321′″ has one or more piercing elements with endssufficiently sharp to cut open sealing material 320′″. Illustratively,seal piercer 321′″ has first piercing element 381′″ having a piercingend disposed adjacent sample preparation reservoir 317′″, secondpiercing element 382′″ having a piercing end disposed adjacent washreservoir 318′″, and third piercing element 383′″ having a piercing enddisposed adjacent substrate reservoir 319′″.

Seal piercer 321′″ also may include slots 638 and 640 configured toreceive a portion of posts 610 and 612 respectively. Accordingly, sealpiercer 321′″ may move within housing 304′″ while the portions of posts610 and 612 remain in slots 638 and 640. Cartridge 300′″ also mayinclude one or more ramps configured to deflect the one or more piercingelements toward sealing material 320′″ to pierce sealing material 320′″.The one or more ramps may be directly coupled to housing 304′″ ofcartridge 300′″. The peak(s) of the one or more ramps may be positionedsuch that the one or more piercing elements travel past the peak(s) whenthe sample collection device is fully inserted in input tunnel 301′″ tofacilitate venting of the reservoirs. Illustratively, cartridge 300′″has first ramp 642 disposed adjacent sample preparation reservoir 317′″,second ramp 644 disposed adjacent wash reservoir 318′″, and third ramp646 disposed adjacent substrate reservoir 319′″. The distance betweeneach ramp and each piercing element in the pre-venting position may bedifferent such that the reservoirs are pierced sequentially. Each rampmay have a break in the middle to fit the portion of seal piercer 321′″adjacent the piercing elements in the venting and mixing positions, asshown in FIG. 15B.

As sample collection device 200′″ is moved distally through input tunnel301′″, preferably seal piercer 321′″ does not move within the cartridgehousing, and remains in a pre-vent position, until sample collectiondevice 200′″ securely engages seal piercer 321′″. Seal piercer 321′″ maysecurely engage sample collection device 200′″ by temporarily orpermanently coupling engager 380′″ of seal piercer 321′″ to samplecollection device 200′″, e.g., at shoulder 220, once sample collectiondevice 200′″ is sufficiently inserted in input tunnel 301′″.

As collector pushes sample collection device 200′″ distally, sealpiercer 321′″ is moved from the pre-venting position towards the ventingposition. Seal piercer 321′″ may move within the cartridge by, forexample, application of a force greater than a threshold force by samplecollection device 200′″ (e.g., at shoulder 220 which may be at thedistal end of shroud 213′″ and/or at the distal end of engagement zone209′″) on seal piercer 321′″ (e.g., at engager 380′″). As samplecollection device 200′″ is moved distally, the piercing element theshortest distance from its ramp first pierces the sealing material abovethat reservoir. For example, as sample collection device 200′″ is moveddistally, seal piercer 321′″ moves generally parallel to movement ofsample collection device 200′″ until second piercing element 382′″contacts second ramp 644 and second ramp 644 deflects second piercingelement 382′″ downwardly to pierce sealing material 320′″ over washreservoir 318′″. As seal piercer 321′″ continues to move distally secondpiercing element 382′″ moves past the peak of second ramp 644 and movesupward out of the hole pierced above wash reservoir 318′″ and thirdpiercing element 383′″ contacts third ramp 646 and third ramp 646deflects third piercing element 383′″ downwardly to pierce sealingmaterial 320′″ over substrate reservoir 319′″. As seal piercer 321′″continues to move distally third piercing element 383′″ moves past thepeak of third ramp 646 and moves upward out of the hole pierced abovesubstrate reservoir 319′″ and first piercing element 381′″ contactsfirst ramp 642 and first ramp 642 deflects first piercing element 381′″downwardly to pierce sealing material 320′″ over sample preparationreservoir 317′″. As seal piercer 321′″ continues to move distally firstpiercing element 381′″ moves past the peak of first ramp 642 and movesupward out of the hole pierced above sample preparation reservoir 317′″.As will be understood to one skilled in the art, the order of piercingmay be varied for sequential piercing.

Advantageously, a configuration where insertion of sample collectiondevice 200′″ causes sample preparation reservoir 317′″, wash reservoir318′″, and/or substrate reservoir 319′″ to be vented sequentiallyensures that the reservoir(s) remain fluidicly sealed prior to samplecollection device 200 insertion, eases resistive forces during insertionof the sample collection device, and facilitates drainage of thereservoir(s) into the analysis channel when the outlet of the respectivereservoir permits fluid flow therethrough.

Referring now to FIGS. 16A through 16J, a configuration for collectingand analyzing a sample using cartridge 300′″ is described.Illustratively, sample collection device 200′″ is inserted in cartridge300′″, although it should be understood that any sample collectiondevice described herein may be used as cartridge 300′″ is designed foruniversal application for different types of sample and different typesof indications, although certain internal components may be substitutedsuch as the shuttle, reagent ball(s), and/or collet based on theapplication. Sample collection device 200′″ is described above withrespect to FIG. 13B. Sample collection device 200′″ illustrativelyincludes engagement zone 209′″ having a plurality of grooves andprotrusions distal to, and spaced apart from, the sealing zone at theproximal portion of sample collection device 200′″. The plurality ofgrooves and protrusions are configured for engagement with one or morecomponents of cartridge device 300′″ for irretractability of samplecollection device 200′″ during partial and full insertion of samplecollection device 200′″ within cartridge 300′″. Engagement zone 209′″may facilitate fixed engagement between sample collection device 200′″and the cartridge device, e.g., via locking members 387′″, such thatsample collection device 200′″ is mated irreversibly with the cartridgewhen sample collection device 200′″ is partially inserted apredetermined distance in the input tunnel of the cartridge, e.g., whenthe distal-most groove of engagement zone 209′″ engages locking members387′″. Sample collection device 200′″ continues to be mated irreversiblywith the cartridge as insertion continues toward the full insertionposition. For example, locking members 387′″ may sequentially engagegrooves of the multiplicity of grooves in engagement zone 209′″ in adistal to proximal direction as sample collection device 200′″ is moveddistally in input tunnel 301′″. Locking members 387′″ may engage theproximal-most groove of the multiplicity of grooves in engagement zone209′″ when sample collection device 200′″ is fully inserted in inputtunnel 301′″. Preferably, sample collection device 200′″ cannot beretracted after both partial and full insertion, thereby reducing therisk of contamination. Engagement zone 209′″ may be configured to becoupled, permanently or temporarily, to the seal piercer of thecartridge device to move the seal piercer within the cartridge deviceresponsive to movement of sample collection device 200′″. In addition oralternatively, engagement zone 209′″ may be configured to activate acontact switch during sample collection device insertion. For example,shoulder 220 at the distal end of engagement zone 209′″ may beconfigured to be coupled to the seal piercer and/or to activate thecontact switch.

Prior to insertion within input tunnel 301′″ of cartridge device 300′″,sample collection device 200′″ is exposed to a sample, e.g., within aninner cheek, the throat, the mouth, a nasal passageway, an ear, fromurine, from blood, from plasma, from saliva, etc. Wicking portion 211′″of sample collection device 200′″ is designed to retain some of thesample to permit analysis of the presence, absence, and/or quantity ofone or more target analytes within the sample using cartridge device300′″ and the reader device. Cartridge device 300′″ may be electricallycoupled to the reader device before or after sample collection device200′″ is inserted in cartridge device 300′″.

Referring to FIG. 16A, the distal end of sample collection device 200′″is first inserted into aperture 302′″ defining the opening of inputtunnel 301′″. As shown, seal piercer 321′″ is in the pre-ventingposition and has not yet pierced seal material 320′″ over thereservoirs, the proximal end of shuttle 324′″ is disposed within thedistal end of collet 618 while the distal end of shuttle 324′″ forms awall of sample preparation reservoir 317′″, and deflector portion 636 ofcollet 618 is in the pre-deflection position where deflector portion 636does not activate contact switch 389′″. In this pre-mixing position,reagent ball 375′″ housed by shuttle 324′″ is within input tunnel 301′″and not exposed to fluid within sample preparation reservoir 317′″.

Referring to FIG. 16B, as sample collection device 200′″ moves distallyin input tunnel 301′″, the distal end of sample collection device 200′″travels through lumen 624 of collet 618 and contacts shuttle 324′″. Forexample, the distal end and/or distal sealing zone 208′″ may contactsecond end 371′″ (e.g., at cavity 395′″ and/or proximal flange 394′″) asillustrated. As explained above with respect to FIGS. 12A through 12E,cavity 395′″ may be sized slightly larger than the outer surface ofwicking portion 211′″ such that the distal end of wicking portion 211′″fits snuggly within cavity 395′″ and distal sealing zone 208′″ may beconfigured to fluidicly seal sample collection device 200′″ to shuttle324′″ such that fluid expelled from sample collection device 200′″travels into sample compartment 370′″. In this pre-mixing position,sample compartment 370′″ is within input tunnel 301′″ and not exposed tofluid within sample preparation reservoir 317′″.

As shown in FIG. 16C, as sample collection device 200′″ moves furtherdistally in input tunnel 301′″, sample collection device 200′″ maydeflect one or more locking members 387′″ of cartridge device 300′″,e.g., via shoulder 220, and sample collection device 200′″ may expelcollected fluid sample into sample compartment 370′″ of shuttle 324′″,e.g., through opening 372′″ of second end 371′″. As is explained above,as sample collection device 200′″ is moved distally, wicking portion211′″ may be compressed to expel collected sample and the distal end ofwicking portion 211′″ may remain substantially in position duringcompression. Intermediate sealing zone 212′″ and/or shroud 213′″ maymove distally in proportion to movement of the handle of samplecollection device 200′″ during such compression. Preferably, as wickingportion 211′″ is compressed and sample is expelled therefrom, theexpelled sample travels through opening 372′″ into sample compartment370′″. During compression, shuttle 324′″ may remain substantially inposition within input tunnel 301′″ and first end 366′″ of shuttle 324′″may continue to form a wall of sample preparation reservoir 317′″ toseal fluid within sample preparation reservoir 317′″. Collet 618 mayhold shuttle 324′″ in the pre-mixing position during compression ofwicking portion 211′″. For example, first and second locking arms ofcollet 618 may hold shuttle 324′″ in the pre-mixing position.

Cartridge device 300′″ may include overflow compartment 397′″ andoverflow lumen 398′″ configured to receive excess sample if the amountof sample introduced into sample compartment 370′″ exceeds apredetermined volume, e.g., exceeds about 20 μl, as explained above.Overflow compartment 397′″ is illustratively formed as part of theinternal component and is sealed on the top surface by the cartridgehousing.

As shown in FIG. 16D, as sample collection device 200′″ moves furtherdistally in input tunnel 301′″, sample collection device 200′″ maydeflect one or more locking members 387′″ of cartridge device 300′″ pastshoulder 220 such that one or more locking members 387′″ lock toengagement zone 209′″ during partial sample collection device 200′″insertion. Illustratively, locking members 387′″ engage the distal-mostgroove of engagement zone 209′″ to accomplish the first lock duringinsertion. As the distal end of shroud 213′″, e.g., at shoulder 220,passes desiccant 325′″, a liquid safety barrier is formed in inputtunnel 301′″ by shroud 213′″ and desiccant 325′″. During wicking portion211′″ compression shown in FIG. 16D, like FIG. 16C, sealing zone 212′″and/or shroud 213′″ may move distally to further expel sample intosample compartment 370′″ while shuttle 324′″ may remain substantially inposition within input tunnel 301′″.

Referring to FIG. 16E, as sample collection device 200′″ moves furtherdistally in input tunnel 301′″, sample collection device 200′″ continuesto deflect one or more locking members 387′″ of cartridge device 300′″past protrusions and into grooves of engagement zone 209′″ in a distalto proximal direction such that one or more locking members 387′″continue to lock to engagement zone 209′″ throughout partial samplecollection device 200′″ insertion. During wicking portion 211′″compression shown in FIG. 16E, like FIGS. 16C and 16D, sealing zone212′″ and/or shroud 213′″ may move distally to further expel sample intosample compartment 370′″ while shuttle 324′″ may remain substantially inposition within input tunnel 301′″. Once sample collection device 200′″causes collet 618 to decouple from shuttle 324′″, shuttle 324′″ may movewithin input tunnel 301′″ from the pre-mixing position to the mixingposition. For example, sample collection device 200′″ may apply a forceon collet 618 during insertion of sample collection device 200′″ ininput tunnel 301′″ to decouple collet 618 from shuttle 324′″. In FIG.16E, engagement zone 209′″, e.g., at shoulder 220, contacts collet 618,e.g., at first and second locking arms.

FIG. 16F is a top view of FIG. 16E showing partial insertion of samplecollection device 200′″ in cartridge 300′″. Collet 618 is disposed ininput tunnel 301′″ and configured to be coupled to shuttle 324′″ in thepre-mixing position. Collet 618 also may be configured to be coupled toshuttle 324′″ during compression of sample collection device 200′″ toallow the distal portion of sample collection device 200′″ to compresswhile shuttle 324′″ remains in place. Illustratively, second end 371′″of shuttle 324′″ is disposed in the lumen of collet 618. In thisexample, collet 618 includes first locking arm 628 and second lockingarm 630 at opposing lateral sides of collet 618. The protrusion of firstlocking arm 628 and protrusion 634 of second locking arm 630 holdproximal flange 394′″ of shuttle 324′″ during compression of the distalportion of sample collection device 200′″. First and second locking arms628 and 630 also may decouple from shuttle 324′″ during insertion ofsample collection device 200′″ in input tunnel 301′″. First and secondlocking arms 628 and 630 may be deflected to decouple first and secondlocking arms 628 and 630 from shuttle 324′″ responsive to a forceapplied on first and second locking arms 628 and 630 by samplecollection device 200′″ during insertion of sample collection device200′″ in input tunnel 301′″. In FIG. 16F, shoulder 220 of samplecollection device 200′″ contacts the ramp of first locking arm 628 andramp 632 of second locking arm 630 during partial sample collectiondevice 200′″ insertion.

Referring to FIG. 16G, as sample collection device 200′″ moves furtherdistally in input tunnel 301′″, sample collection device 200′″, e.g., atshoulder 220, applies a force on the ramps of first and second lockingarms 628 and 630 to cause the protrusions of first locking arm 628 andprotrusion 634 of second locking arm 630 to deflect outwardly. The rampsare shaped such that the protrusions decouple from proximal flange 394′″of shuttle 324′″ to unlock shuttle 324′″ from collet 618 and permitshuttle 324′″ to move from the pre-mixing position towards the mixingposition.

As depicted in FIGS. 16F and 16G, sample preparation reservoir 317′″,wash reservoir 318′″, and/or substrate reservoir 319′″ may each havesymmetric shapes. For example, each of the walls of sample preparationreservoir 317′″ may form a symmetric shape wherein each of the walls ofsample preparation reservoir 317′″ meet at an angle greater than apredetermined angle, e.g., 60°, 90°, to facilitate fluid emptyingthrough the outlet of sample preparation reservoir 317′″. Sonicatorelement 327′″ may be positioned off-center as illustrated of samplepreparation reservoir 317′″ to facilitate mixing of the fluid withinsample preparation reservoir 317′″. Similarly, each of the walls of washreservoir 318′″ and/or substrate reservoir 319′″ may form a symmetricshape wherein each of the walls of wash reservoir 318′″ and/or substratereservoir 319′″ meet at an angle greater than a predetermined angle,e.g., 60°, 90°, to facilitate fluid emptying through the respectiveoutlets of wash reservoir 318′″ and/or substrate reservoir 319′″.

Referring to FIG. 16H, as sample collection device 200′″ moves furtherdistally in input tunnel 301′″, sample collection device 200′″ contactsseal piercer 321′″. For example, shoulder 220 of sample collectiondevice 200′″ may contact engager 380′″ of seal piercer 321′″ withininput tunnel 301′″. As collector pushes sample collection device 200′″distally from the pre-mixing position towards the mixing position,shuttle 324′″ may be partially moved within sample preparation reservoir317′″ as shown in FIG. 16H. As shuttle 324′″ moves distally, first end366′″ may unseal such that one or more reagent balls 375′″ withinshuttle 324′″ and the collected sample within shuttle 324′″ are exposedto fluid within sample preparation reservoir 317′″. Advantageously,before first end 366′″ of shuttle 324′″ is unsealed, second end 371′″ ofshuttle 324′″ is fluidicly sealed by sample collection device 200′″ suchthat fluid from sample preparation reservoir 317′″ does not leak intoinput tunnel 301′″ beyond second end 371′″ and/or intermediate sealingzone 212′″. In addition, as distal movement of sample collection device200′″ causes the transition from the pre-mixing position toward themixing position, sealing members 614 of shuttle 324′″ continuouslyfluidicly seal the fluid in sample preparation reservoir 317′″ frominput tunnel 301′″.

Referring to FIG. 16I, as sample collection device 200′″ moves furtherdistally in input tunnel 301′″, sample collection device 200′″ movesseal piercer from the pre-venting position towards the venting position.As collector pushes sample collection device 200′″ distally, samplecollection device 200′″ applies a force to seal piercer 321′″ (e.g., viacontact between shoulder 220 and engager 380′″) to cause the piercingelements to move to sequentially pierce sealing material 320′″ over thereservoirs as described above with respect to FIGS. 15A through 15D. Inaddition, as collector pushes sample collection device 200′″ moredistally from the pre-mixing position toward the mixing position,shuttle 324′″ moves more distally within sample preparation reservoir317′″. Sample collection device 200′″ also may contact collet 618 tofacilitate activation of contact switch 389′″. For example, shoulder 220of sample collection device 200′″ may contact deflection portion 636 ofcollet 618.

As sample collection device 200′″ moves further distally in input tunnel301′″, sample collection device 200′″ and cartridge device 300′″ aremoved into the mixing, venting, analysis positions shown in FIG. 16Jwherein the expelled sample in sample compartment 370′″ and reagent ball375′″ are mixed within fluid of sample preparation reservoir 317′″. InFIG. 16J, sample collection device 200′″ is fully inserted in inputtunnel 301′″. As shown, seal piercer 321′″ is in the venting positionand has pierced seal material 320′″ over each of the reservoirs, shuttle324′″ has moved distally from collet 618 such that the sample andreagent ball 375′″ are mixing with the fluid in sample preparationreservoir 317′″, deflector portion 636 of collet 618 is in thedeflection position where deflector portion 636 has activated contactswitch 389′″, and locking members 387′″ have locked sample collectiondevice 200′″ in input tunnel 301′″.

In this mixing position, first end 366′″ of shuttle 324′″, the one ormore reagent ball compartments of shuttle housing one or more reagentballs, and/or the one or more sample compartments housing the sample(s)to be analyzed may be disposed within sample preparation reservoir317′″. As explained above, the components fluidicly seal samplepreparation reservoir 317′″ in the mixing position such that thecollected sample(s), the reagent ball(s), and the fluid within samplepreparation reservoir 317′″ are sealed within the reservoir and may bemixed within sample preparation reservoir 317′″.

Sample collection device 200′″ may be configured to activate contactswitch 389′″ of circuit board 331′″. For example, insertion of samplecollection device 200′″ may cause collet 618 to activate contact switch389′″. Illustratively, deflector portion 636 of collet 618 deflects toactivate contact switch 389′″ responsive to a force applied on deflectorportion 636 by sample collection device 200′″ during insertion of samplecollection device 200′″ in input tunnel 301′″. Shoulder 220 of samplecollection device 200′″ may contact deflector portion 636 and forcedeflector portion 636 downwardly as sample collection device 200′″ movesdistally in input tunnel 301′″ to activate contact switch 389′″.Depression of contact switch 389′″ may complete a circuit such thatelectrical signals may be transmitted, e.g., to the reader device and/orthe computing device running the software-based user interface system.In this manner, proper insertion of sample collection device 200′″within the input tunnel generates an electrical signal that may betransmitted to the reader device and/or computing device to notify thereader device and/or computing device of such proper insertion. Whilecontact switch 389′″ is positioned within input tunnel 301′″ such thatdepression of contact switch 389′″ indicates full insertion of samplecollection device 200′″ at the mixing position, contact switch 389′″also could be positioned within input tunnel 301′″ to indicate partialinsertion of sample collection device 200′″. In addition, oralternatively, more than one contact switch may be disposed in the inputtunnel such that insertion of the sample collection device within thetunnel may be tracked by sequential depression of the contact switchesaligned along the input tunnel.

In the mixing position, locking members 387′″ are configured toirreversibly lock sample collection device 200′″ within cartridge device300′″. Locking members 387′″ may engage the proximal-most groove of themultiplicity of grooves in engagement zone 209′″ when sample collectiondevice 200′″ is fully inserted in input tunnel 301′″ as shown in FIG.16J. Locking members 387′″ may be biased inwardly in input tunnel 301′″such that a locking end of locking member 387′″ engages samplecollection device 200′″ in the mixing position. Advantageously, lockingsample collection device 200′″ (e.g., longitudinally and/or axially)within input tunnel 301′″ during partial insertion and full insertionpromotes sealing of sample preparation reservoir 317′″ over time assample collection device 200′″ cannot be retracted once locked tofacilitate safe disposability and consistency of testing because a usercannot pull out sample collection device 200′″ from cartridge device300′″ inadvertently after partial or full insertion. In addition, in themixing position, proximal sealing zone 207′″ of sample collection device200′″ is configured to seal input tunnel 301′″ at aperture 302′″. Inthis manner, proximal sealing zone 207′″ provides additional structureto minimize or eliminate fluid leakage from cartridge device 300′″,e.g., at aperture 302′″. Proximal sealing zone 207′″ of samplecollection device 200′″ is also configured to contact the cartridgehousing to resist insertion force by collector once sample collectiondevice 200′″ and cartridge device 300′″ are properly in the mixingposition.

In the mixing position, sonicator element 327′″ may be used to enhancemixing the fluid in sample preparation reservoir 317′″ with the sampleand reagent ball(s) as described above with respect to FIGS. 10N through10P.

Referring now to FIGS. 17A through 17D, sonicator element 327′″ may beelectrically coupled to circuit board 331′″ via first spring contact392′″ and second spring contact 392′″ as shown. Preferably, sonicatorelement 327′″ is electrically coupled to circuit board 331′″ only viafirst and second spring contacts 392′″, e.g., without a wired connectionbetween sonicator element 327′″ and circuit board 331′″. Beneficially,spring contacts 392′″ absorb movement of sonicator element 327′″ suchthat circuit board 331′″ vibrates minimally in a suitable manner whensonicator element 327′″ is activated, e.g., responsive to signalstransmitted by the reader device, and spring contact 392′″ permits easeof assembly and reproducibility compared to soldering which mayadversely impact sonicator element 327′″. Sonicator element 327′″ mayform a wall, e.g., part of the bottom wall, of sample preparationreservoir 317′″. Sonicator element 327′″ may be adhered to internalcomponent 316′″ to form the wall. For example, an annulus of adhesive,e.g., epoxy, may be applied on the top surface of sonicator element327′″ and UV cured to internal component 316′″ at the bottom of samplepreparation reservoir 327′″. Such an annulus of adhesive permits fluidicsealing between sonicator element 327′″ and sample preparation reservoir317′″ while permitting sonicator element 327′″ to vibrate duringactivation. As is explained above, sonicator element 327′″ may bepositioned off-center of sample preparation reservoir 317′″ tofacilitate mixing of the fluid with the sample and reagent ball(s)within sample preparation reservoir 317′″.

Referring now to FIGS. 18A and 18B, an alternative cartridge foranalyzing a sample is described. Cartridge device 300″″ may beconstructed similarly to cartridge device 300′″, wherein like componentsare identified by like-primed reference numbers, except cartridge device300″″ includes collet 618′ and shuttle 324″″ which may be constructedsimilarly to shuttle 324 of FIG. 9A. In this embodiment, cartridgedevice 300″″ is designed for relatively small sample collection, e.g.,less than 10 microliters, preferably 2-5 microliters, of a sample, e.g.,from a nasal passageway, from an ear, from blood. Cartridge device 300″″may be further fitted with a reagent ball(s) intended to identifydifferent target analytes that may be indicative of, for example,inflammation, influenza, testosterone, fertility, HIV, or Vitamin D.FIGS. 18A and 18B illustrate the ease of interchangeability betweencartridge device 300′″ and cartridge device 300″″.

As shown in FIG. 18A, collet 618′ has one or more protrusions 637disposed the distal end of collet 618′ configured to contact second end371″″ of shuttle 324″″ in the pre-mixing position. The one or moreprotrusions 637 may contact a sealing member, e.g., O-ring, at secondend 371″″ of shuttle 324″″ to retain the sealing member in position. Theone or more protrusions may have lead-in angles configured to guide thedistal portion of the sample collection device into the opening atsecond end 371″″ of shuttle 324″″. Similar to that described above,collet 618′ is configured to decouple from shuttle 324″″ responsive to aforce applied by the sample collection device on shuttle 324″″, e.g.,via the shoulder of the distal sealing zone on second end 371″″ ofshuttle 324″″, during sample collection device insertion in the inputtunnel. In this manner, shuttle 324″″ moves from the pre-mixing positionto the mixing position as described above.

Referring now to FIG. 19, a process for monitoring temperature duringenhanced mixing via the sonication element is described. As is describedbelow, the computerized reader may largely control the operations of thedetection system. The reader includes a processor with memory, thememory having instructions stored thereon for implementing variousmethods needed to successfully detect the presence, absence, and/orquantity of one or more target analytes within a collected sample. Forexample, the computerized reader may cause the process of FIG. 19 to beperformed in an automated manner.

As is described above, a sonciator element (e.g., sonicator element 327,327′, etc.) of the cartridge may be activated for enhanced mixing of thecontents in the sample preparation reservoir when the sample collectiondevice and the cartridge are in the mixing position. At 702, the processbegins. The process may begin responsive to an event. For example, theprocess may begin when the processor of the reader device receives asignal indicating that a contact switch has been activated in thecartridge device responsive to full insertion of the sample collectiondevice in the cartridge device.

At 704, the sonicator element, e.g., a piezoelectric transducer, isactivated. Upon activation, the sonicator element emits acoustic wavestoward the sample preparation reservoir to move fluid within thereservoir. The sonicator element may emit acoustic waves at apredetermined frequency, e.g., 4 MHz, which may be modified by thereader. As is described above, the acoustic waves may cause fluid withinthe reservoir to move in a wave pattern to mix the fluid in thereservoir. The processor of the reader device may activate the sonicatorelement by causing transmission of electrical signals to the sonicatorelement, e.g., via one or more electrical leads of the printed circuitboard in the cartridge.

At 706, a temperature is sampled. The temperature may be indicative oftemperature of the fluid within the sample preparation reservoir. Forexample, the cartridge may include a temperature sensor, e.g.,temperature sensor 616 disposed on the circuit board, configuredtogenerate a signal indicative of temperature of the fluid in the samplepreparation reservoir. The signal may be transmitted from the cartridgeto the reader, e.g., via one or more electrical leads on the printedcircuit board of the cartridge, when the cartridge and reader areelectrically coupled. The signal may be processed by the processor ofthe reader device to determine whether the signal indicates atemperature of the fluid in the reservoir outside a threshold.

At 708, it is determined whether the sampled temperature is too high.For example, the processor of the reader device may compare thetemperature information from the signal generated by the temperaturesensor to temperature thresholds stored in memory of the reader device.For example, the memory may store a threshold high temperature, e.g.,above 42° C., and/or a look-up table with threshold high temperaturesbased on cartridge specific parameters such that the processor maycompare the sensed temperature to the stored information to determinewhether a sensed temperature is too high. If the temperature isdetermined to be outside a threshold, e.g., too high, too low, notwithin a range, emission of acoustic waves from the sonicator elementmay be modified.

At 710, if the temperature is determined to be too high, emission ofacoustic waves from the sonicator element may be modified by loweringthe duty cycle of the sonicator element. For example, the processor ofthe reader device may modify emission of the acoustic waves from thesonicator element by transmitting electrical signals to the sonicatorelement to cause the sonicator element to lower the duty cycle. Theprocessor may modify emission of the acoustic waves from the sonicatorby deactivating the sonicator if the signal indicates the temperature ofthe fluid in the reservoir is above the threshold.

At 712, it is determined whether the sampled temperature is too low. Forexample, the processor of the reader device may compare the temperatureinformation from the signal generated by the temperature sensor totemperature thresholds stored in memory of the reader device. Forexample, the memory may store a threshold low temperature, e.g., below37° C., and/or a look-up table with threshold low temperatures based oncartridge specific parameters such that the processor may compare thesensed temperature to the stored information to determine whether asensed temperature is too low.

At 714, if the temperature is determined to be too low, emission ofacoustic waves from the sonicator element may be modified by raising theduty cycle of the sonicator element. For example, the processor of thereader device may modify emission of the acoustic waves from thesonicator element by transmitting electrical signals to the sonicatorelement to cause the sonicator element to increase the duty cycle.

At 716, it is determined whether the temperature indicative oftemperature of the fluid within the sample preparation reservoir iswithin a threshold range, e.g., below the threshold high temperature andabove the threshold low temperature, for a predetermined amount of time(e.g., between 3 minutes to 15 minutes, between 3 minutes to 10 minutes,between 3 minutes to 5 minutes, about 10 minutes), which may becumulative or consecutive, such that the sonicator element may betimed-out. For example, the memory of the reader may store thepredetermined amount of time that the temperature indicative of fluidtemperature within the sample preparation reservoir should be within thethreshold temperature range. The predetermined amount of time may be thetime required for suitable mixing of the fluid within the samplepreparation reservoir with the sample and the reagent ball(s) forisothermal amplification for analysis. The acoustic emissions from thesonicator element may both heat fluid within the sample preparationreservoir and mix the contents of the sample preparation reservoir atthe macro and the micro level for isothermal amplification. For example,the sonicator element may pass energy into the sample preparationreservoir sufficient to increase the temperature in the samplepreparation reservoir such that an amplification reaction may occur suchas isothermal DNA or RNA amplification, thereby generating nucleic acidamplicons for downstream detection. Such downstream detection may bepartially based on the binding of nucleic acids or detectable moietiesthereon or therein to affinity molecules on magnetic particles (whichcould be in a combination of anitbodies, DNA probes, detectablemoieties, and/or enzymes) or it could be based on specific binding tosurface bound affinity molecules on one or more working electrodes eachwith their own population of affinity molecules. Alternatively, oradditionally, such a reaction may take place in the sample preparationreservoir and be observed there. If the processor of the readerdetermines that the sampled temperature has not been within thethreshold range for the predetermined amount of time, the processreturns to 706.

At 718, the sonicator element is deactivated. For example, the sonicatorelement may be deactivated if the sampled temperature has been withinthe threshold range for the predetermined amount of time. The processorof the reader may determine that the sampled temperature has been withinthe threshold range for the predetermined amount of time.

At 720, the process ends after the sonicator element is deactivated.

Referring now to FIG. 20, a graph showing temperature in Celsius versustime in seconds is shown. The graph shows the measured temperature ofthe fluid mixing in the sample preparation reservoir at line 730, themeasured temperature at the thermistor, e.g., temperature sensor 616, atline 732, the measured ambient temperature at line 734, and the measuredtemperature at the piezoelectric transducer, e.g., sonicator element327, 327′, etc., at line 736. As will be observed, the duty cycle of thesonicator is reduced at about the 40 second mark on the graph, causingthe temperature of the fluid mixed in the sample preparation reservoirto decrease. The duty cycle of the sonicator is again reduced around the520 second mark on the graph, again causing the temperature of the fluidmixed in the sample preparation reservoir to decrease.

The Reader Device

The reader device, or reader, of various embodiments is, comprises, oris comprised of, a specialized computer. The computer includes aprocessor with memory having instructions stored thereon for executingone or more methods for detecting the presence, absence, and/or quantityof one or more target analytes in a sample. In various embodiments, thereader's computer controls the operations of the detection system,controlling when and how various functions of the system occur, such as,for example: mixing of the fluids in the sample preparation reservoir ofthe cartridge, opening of valves, and/or localization of magneticparticles over the sensors. To control such operations, the computerizedreader is configured to receive information from, and send informationto, physical components present within the reader or cartridge.

FIG. 21A illustrates a perspective view of an exemplary reader deviceand FIG. 21B is an exploded view illustrating the internal components ofthe reader device of FIG. 21A. Reader device 400 may include opening 401in cartridge dock 402, housing 403, user interface 404, power supply405, first magnetic generator 406, second magnetic generator 407,magnetic generator housing 408, elastic member 409, light pipe 410,circuit board 411, processor 412, communication circuit 413, electricalconnector 414, inductive coil 415, alignment magnets 416, and/or base417.

Opening 401 of reader device 400 permits the cartridge device to bedocked within cartridge dock 402. The cartridge, when received by readerdevice 400, may be disposed on or in, partially or fully, or otherwisecoupled to, reader device 400. Several of the reader components may bestrategically positioned in particular locations relative to cartridgedock 402 to achieve desired interactions with the cartridge. Forexample, electrical connector 414 may be positioned relative tocartridge dock 402 such that the electrical connector of the cartridgedevice is electrically coupled to electrical connector 414 when thecartridge device is inserted in cartridge dock 402. In addition,magnetic field generators 406 and 407 may be positioned relative tocartridge dock 402 such that magnetic field generators 406 and 407 aredisposed under the working electrode of the cartridge device when thecartridge device is inserted in cartridge dock 402.

Housing 403 is configured to house the internal components of readerdevice 400 and may cooperate with cartridge dock 402 and base 417 tohouse the internal components.

User interface 404 may be used to receive inputs from, and provideoutputs to, a user. Illustratively, user interface 404 includes LEDsconfigured to notify a user when a cartridge device is properly insertedinto reader device 400 and/or when a sample collection device isproperly inserted in the cartridge device. User interface 404 may becoupled to processor 412. User interface 404 may include a touchscreen,LED matrix, other LED indicators, or other input/output devices forreceiving inputs from, and providing outputs to, a user. In otherembodiments, user interface 404 is not present on reader 400, but isinstead provided on a remote computing device communicatively connectedto reader 400 via the communication circuit 413. User interface also maybe a combination of elements on the reader and a remote computingdevice.

Power supply 405 may be a suitable battery such as a replaceable batteryor rechargeable battery and apparatus may include circuitry for chargingthe rechargeable battery, and a detachable power cord. Power supply 405may be charged by charger 500 via an inductive coil within the chargerand inductive coil 415. Alternatively, the power supply may be a port toallow reader device 400 to be plugged into a conventional wall socket,e.g., via a cord with an AC to DC power converter, for poweringcomponents within the housing.

Magnetic field generators 406 and 407 may be inductors or otherelectromagnetic components movably affixed within reader 400. Magneticfield generators 406 and 407 may be permanent magnets. Magnetic fieldgenerators 406 and 407 are positioned such that, when a cartridge iselectrically coupled to reader device 400, the working electrode isdisposed directly within a magnetic field created by magnetic fieldgenerators 406 and 407. In various embodiments, the magnetic field(s)are the cause of localization; the magnetic field(s) are what inducemagnetic particles and accompanying hybridized molecules to localizewithin the analysis zone. First and second magnetic generators 406 and407 may be configured to generate a magnetic field over the length of asingle working electrode to promote homogenous distribution of aplurality of magnetic particles over the length of the single workingelectrode.

Magnetic field generators 406 and 407 emit a magnetic field sufficientlystrong to cause the magnetic particles released into the analysischannel from the sample preparation reservoir to remain localized overmagnetic field generators 406 and 407, and thereby over the workingelectrode, as the wash solution and/or the fluid carrying chemicalsubstrates flows over the magnetic particles in the analysis channel inthe cartridge.

Magnetic generator housing 408 is configured to house magnetic fieldgenerators 406 and 407. Magnetic generator housing 408 may be coupled toelastic member 409 that permits movement of magnetic field generators406 and 407, e.g., upon insertion and removal of a cartridge device fromcartridge dock 402.

Reader device 400 may include light pipe 410 designed to guide lightfrom LEDs within reader device 400 to user interface 404.

Circuit board 411 includes electrical components and permitselectrically coupling between processor 412, communication circuit 413,and/or electrical connector 414. One or more electrical componentsand/or circuits may perform some of or all the roles of the variouscomponents described herein. Although described separately, it is to beappreciated that electrical components need not be separate structuralelements. For example, processor 412 and communication circuit 413 maybe embodied in a single chip. In addition, while processor 412 isdescribed as having memory, a memory chip(s) may be separately provided.

Processor 412 may be a general purpose processor, a digital signalprocessor (DSP), an application specific integrated circuit (ASIC), afield programmable gate array (FPGA) or other programmable logic device,discrete gate or transistor logic, discrete hardware components, or anysuitable combination thereof designed to perform the functions describedherein. A processor may also be implemented as a combination ofcomputing devices, e.g., a combination of a DSP and a microprocessor, aplurality of microprocessors, one or more microprocessors in conjunctionwith a DSP core, or any other such configuration.

Processor 412 may contain memory and/or be coupled, via one or morebuses, to read information from, or write information to, memory. Thememory may include processor cache, including a multi-level hierarchicalcache in which different levels have different capacities and accessspeeds. The memory may also include random access memory (RAM), othervolatile storage devices, or non-volatile storage devices. The storagedevices can include, for example, hard drives, optical discs, flashmemory, and Zip drives.

Processor 412, in conjunction with firmware/software stored in thememory may execute an operating system, such as, for example, Windows,Mac OS, Unix or Solaris 5.10. Processor 412 also executes softwareapplications stored in the memory. In one non-limiting embodiment, thesoftware comprises, for example, Unix Korn shell scripts. In otherembodiments, the software may be programs in any suitable programminglanguage known to those skilled in the art, including, for example, C++,PHP, or Java.

Communication circuit 413 is configured to transmit information, such assignals indicative of the presence, absence, and/or quantity of one ormore target analytes within a sample, locally and/or to a remotelocation such as a server. Communication circuit 413 is configured forwired and/or wireless communication over a network such as the Internet,a telephone network, a Bluetooth network, and/or a WiFi network usingtechniques known in the art. Communication circuit 413 may be acommunication chip known in the art such as a Bluetooth chip and/or aWiFi chip. Communication circuit 413 may include a receiver and atransmitter, or a transceiver, for wirelessly receiving data from, andtransmitting data to a remote computing device. In some suchembodiments, the remote computing device may be a mobile computingdevice that provides the system with a user interface; additionally oralternatively, the remote computing device is a server. In embodimentsconfigured for wireless communication with other devices, communicationcircuit 413 may prepare data generated by processor 412 for transmissionover a communication network according to one or more network standardsand/or demodulates data received over a communication network accordingto one or more network standards.

Processor 412 is also coupled to electrical connector 414, which mayinclude an EDGE card or other electrical connector, to send electricalsignals to, and receive electrical signals from, the circuit boardcomponent of the cartridge (e.g., via electrical connector 312).Electrical connector 414 may be located on, under, within, or adjacentto cartridge dock 402 and is positioned such that the pins of electricalconnector 414 make contact with, and establish electrical connectivitywith, the electrical leads of a docked cartridge device. Electricalconnector 414 thereby establishes electrical continuity between thesensors on the circuit board of the cartridge and electrochemicalcircuitry within the reader. Electrical connector 414 of the reader alsomay establish electrical continuity with one or more heating elements,if present on the circuit board of the cartridge. Reader device 400 mayinclude a portion of an electrochemical circuit, which is completed withthe addition of the cartridge based on electrical continuity betweenelectrical connector 414 and the electrical leads of the cartridge. Theaddition of the cartridge may complete or close the circuit. Couplingthe cartridge to reader 400 may activate reader device 400, causing itto “wake up.” Once awoken, electrical connector 414 may identify signalsbeing received from a portion of the cartridge to identify what type ofcartridge is coupled to its dock. Electrical connector 414 may receivesignals indicative of information on cartridge type (e.g., inflammation,influenza, testosterone, fertility, Vitamin D), cartridge identificationinformation (e.g., serial number), and/or calibration information frommemory within the cartridge and transmit such information to processor412 for processing.

Once awoken, reader device 400 also may determine what test protocol torun for the identified cartridge and/or searches for, and connects to,nearby mobile computing devices.

Reader device 400 may include one or more magnets 416 configured toalign with one or more magnets in the charger to facilitate efficientenergy transfer between inductive coil 415 and an inductive coil withinthe charger. Illustratively, four magnets are used. Magnets 416 may bekeyed to align with a corresponding keyed magnet in the charger.

Referring now to FIGS. 22A through 22D, insertion of a cartridge devicewithin a reader device is described. As shown in the cutaway view ofreader device 400 in FIG. 22A, first and second magnetic generators 406and 407 may extend partially into opening 401 through cartridge dock402. In FIG. 22A, first and second magnetic generators 406 and 407 areshown in a raised position. Elastic member 409 may be biased to causefirst and second magnetic generators 406 and 407 to rest in the raisedposition.

FIG. 22B shows cartridge device 300, having sample collection device 200partially inserted therein, being inserted into reader device 400.Specifically, cartridge device 300 is inserted in opening 401 atcartridge dock 402. As cartridge device 300 moves distally intocartridge dock 402, the top surfaces of first and second magneticgenerators 406 and 407 preferably do not contact electrical connector312 and first contact first ramp portion 313. First ramp portion 313causes first and second magnetic generators 406 and 407 to graduallydepress during further distal insertion, e.g., by causing a downwardforce on first and/or second magnetic generators 406, 407 that depresseselastic member 409. As cartridge device 300 is inserted past first rampportion 313, first and/or second magnetic generators 406, 407 contactbottom surface 308 of cartridge device 300 and move to a depressedposition as shown in FIG. 22B.

As cartridge device 300 moves further distally into cartridge dock 402,first and/or second magnetic generators 406, 407 contact second rampportion 314 which ramps up into magnetic generator depression 315.Second ramp portion 314 gradually guides first and/or second magneticgenerators 406, 407 into magnetic generator depression 315 as shown inFIGS. 22C and 22D. FIGS. 22C and 22D depict cartridge device 300inserted in reader device 400 in the analysis position where first andsecond magnetic generators 406 and 407 are disposed in magneticgenerator depression 315 and electrical connector 312 of cartridgedevice 300 is electrically coupled to electrical connector 414 of readerdevice 400. Magnetic generator depression 315 is disposed beneath one ormore working electrodes of cartridge device 300 such that first andsecond magnetic generators 406 and 407 move up into magnetic generatordepression 315 into a raised position and are disposed adjacent the oneor more working electrodes when cartridge device 300 is fully insertedin the reader. The bias of elastic member 409 may cause first and secondmagnetic generators 406 and 407 to move up into magnetic generatordepression 315. Second ramp portion 314 also facilitates removal ofcartridge device 300 from reader device 400 by gradually depressingfirst and second magnetic generators 406 and 407 during removal ofcartridge device 300.

The interaction of the bias of elastic member 409 and magnetic generatordepression 315 may allow for first and second magnetic generators 406and 407 to be positioned as close to the working electrode as possible.The closer first and second magnetic generators 406 and 407 are to theworking electrode, the more force the magnet field is able to exert,meaning that smaller magnets or inductors are capable of exertingequivalent magnetic field strengths as larger, more costly magnets orinductors. The use of small magnets or inductors is particularlyadvantageous in embodiments having multiple magnetic fields and multipleanalysis zones (for example, in embodiments configured to detect aplurality of different target analytes), because the smaller the magnetor inductor, the less the magnetic fields overlap. Smaller magneticfields can limit the amount of cross talk between the magnets orinductors under the different detection sensors.

Referring to FIGS. 23A and 23B, graphs are shown depicting the measuredstrengths of magnetic fields at varying distances from the analysischannel for designs where one magnetic generator generates a magneticfield for one working electrode (FIG. 23A) and where two magneticgenerators generates a magnetic field for one working electrode (FIG.23B). It has been discovered that magnetic fields at or near theabsolute peak strength will retain suitable magnetic particles in theanalysis channel over the working electrode, but magnetic particles tendto wash off the working electrode as the magnetic field strengthdecreases from the peaks toward zero, creating dead zones 750 and 751.Use of two magnetic field generators creates a much more uniformabsolute magnetic field over the working electrode as compared to use ofone magnetic field generator. Accordingly, dead zone 751 for a dualmagnet design is significantly smaller than dead zone 750 for a singlemagnet design. Accordingly, as described above, first and secondmagnetic generators 406 and 407 may be used in reader device 400 togenerate a magnetic field over the length of a single working electrodeto promote homogenous distribution of a plurality of magnetic particlesover the length of the single working electrode.

The Computerized Methods of Detection

The timing of heat delivery and valve opening within the cartridgedevice may be precisely timed and controlled by the reader device. Forexample, the reader may control when heat-generating current flowsthrough the heating elements. Current may flow from the reader to thecartridge to cause actuation in the following sequence: (1) valveactuation for sample preparation reservoir, (2) fluidic isolatoractuation, if present, (3) valve actuation for wash reservoir, ifpresent, then (4) valve actuation for chemical substrate reservoir.Actuation of each valve may be timed such that: the respective valvefully actuates, the associated reservoir has time to empty its contentsinto the analysis channel, and at least some of the contents of thereservoir have time to travel to the absorbent pad positioned downstreamof the sensors before the contents of the next reservoir is released. Insome embodiments, the time between valve actuations is selected to begreat enough for the absorbent pad to entirely or substantially absorbfluid present within the analysis channel. Advantageously, in suchembodiments, very little mixing occurs between the contents ofsuccessive reservoirs. In addition, or alternatively, actuation of eachvalve may be based on a feedback control system wherein the cartridgeand/or reader recognizes when flow has initiated and/or stopped from therespective reservoirs, e.g., using a flow sensor disposed in theanalysis channel adjacent to the respective reservoir and electricallycoupled to the memory of the cartridge and/or the processor of thereader, such that the valves may be turned on and off with respect tothe “state” of the progression of events, e.g., based on signals sensedby the flow sensors disposed adjacent each reservoir.

As mentioned above, the computerized reader largely controls theoperations of the detection system. The reader includes a processor withmemory, the memory having instructions stored thereon for implementingvarious methods needed to successfully detect the presence, absence,and/or quantity of one or more target analytes within a collectedsample. For example, an embodiment of one method performed by thecomputerized reader in an automated manner is provided in FIG. 24.

At block 802, the computerized reader detects the presence of acartridge loaded into or onto the reader. For example, a cartridge maybe coupled to the reader such that electrical leads on the cartridgecome into physical contact with electrical pins on the reader,completing a circuit that turns on the reader and signals the reader tothe presence of a cartridge.

At block 804, the reader detects identification information associatedwith the cartridge. For example, the cartridge may include cartridgetype information stored within its memory, which generates signalsunique to the particular type of the cartridge, allowing the reader todistinguish between cartridges and types. The cartridge also may includecalibration information stored within its memory.

The reader's processor receives the cartridge type information signals,and as shown at block 806, may identify a proper test protocol for thecartridge based on the cartridge type information. The reader'sprocessor may compare cartridge type information signals to a databaseof protocols based on the cartridge type stored in memory. If theprocessor does not recognize the cartridge type information, theprocessor may communicate with a remote computing device such as amobile computing device and/or a server to signal that an unidentifiablecartridge has been detected. In some embodiments, the reader downloadsupdates directly from a server or indirectly with the mobile computingdevice acting as an intermediary. In some embodiments, when an unknowncartridge type is detected, a user is prompted via the user interface ofthe mobile computing device, to download updates; in other embodiments,the updates are downloaded automatically. In various embodiments, theupdates include newly developed cartridge types and test protocols. Oncethe new types and test protocols are downloaded, they will be added tothe reader's database of supported tests so that future tests with thiscartridge type will automatically be recognized and implemented withoutthe need for communicating with remote computing devices. The reader'sprocessor also may receive calibration information signals and mayaccount for the calibration information during the test protocol.

As shown at block 808, the computerized reader detects insertion of asample collection device into the cartridge. For example, the reader mayreceive electrical signals indicating that the contact switch in thecartridge device has be activated responsive to insertion of the samplecollection device into the input tunnel, which may also substantiallycorrespond to the sample entering the sample preparation reservoir. Thesample (and reagent ball(s), if present) is then mixed within the fluidof the sample preparation reservoir by introducing the sample (andreagent ball(s), if present) into the sample preparation reservoir.

At block 810, the reader's processor sends signals to the sonicatorelement to instruct it to initiate a sonication protocol to mix aplurality of reagents, affinity molecules, and sample particles within afluid disposed within the sample preparation reservoir. In variousembodiments, the resulting mixture includes magnetic particles bound to:target analytes, target analytes and detector agents, and/or competitivebinding agents. As used herein, sandwich complexes refer to magneticparticles bound directly or indirectly to target analytes and detectoragents; competitive binding complexes refer to magnetic particles boundto competitive binding agents. Each sandwich complex and competitivebinding complex may include a detector agent bound within the complex.In one embodiment described here, the detector agent is an oxidizingenzyme. The sonicator also may pass energy into the sample preparationreservoir sufficient to increase the temperature in the samplepreparation reservoir such that an amplification reaction may occur suchas isothermal DNA or RNA amplification, thereby generating nucleic acidamplicons for downstream detection. Such downstream detection may bepartially based on the binding of nucleic acids or detectable moietiesthereon or therein to affinity molecules on magnetic particles (whichcould be in a combination of anitbodies, DNA probes, detectablemoieties, and/or enzymes) or it could be based on specific binding tosurface bound affinity molecules on one or more working electrodes eachwith their own population of affinity molecules. Alternatively, oradditionally, such a reaction may take place in the sample preparationreservoir and be observed there.

As shown at block 812, the reader may monitor temperature of the fluidin the sample preparation reservoir during mixing by the sonicatorelement and isothermal amplification. For example, a temperature sensorin the cartridge may transmit signals indicative of temperature of thefluid in the sample preparation reservoir, as described above withrespect to FIG. 19.

As shown at block 814, the reader may generate a current, which heats orotherwise stimulates a first heating element, thereby causing heat totransfer to a heat-actuated valve sealing the outlet of the samplepreparation reservoir within the cartridge. Such heating may cause thevalve to melt or undergo another phase change, which allows fluid toflow out of the sample preparation reservoir into an analysis channelvia capillary action. As the fluid flows, it transports the mixture withit, and the magnetic particles within the mixture, including magneticparticles within sandwich complexes and/or competitive bindingcomplexes, localize over one or more magnetic fields within the analysischannel, forming one or more localized samples.

Optionally, at block 816, the reader generates a current, which heats orotherwise stimulates a second heating element that causes a fluidicisolator to block the outlet of the sample preparation reservoir fromthe rest of the analysis channel. In this manner, fluid released fromother reservoirs cannot flow into the sample preparation reservoirand/or cause undesirable reactions with leftover reagents.

Optionally, at block 818, the reader generates a current, which heats orotherwise stimulates a third heating element such that a second valvewithin the cartridge undergoes a phase change and a wash solution flowsout of a wash reservoir into the analysis channel. In variousembodiments, the wash solution removes, from the one or more localizedsamples, oxidizing enzymes (or other detector agents) that are notindirectly bound to magnetic particles.

At block 820, the reader generates a current, which heats or otherwisestimulates a fourth heating element such that a third valve within thecartridge undergoes a phase change and a solution of substrates flowsout of a substrate reservoir into the analysis channel. In variousembodiments, when the detector agent is an oxidizing enzyme, theoxidizing enzymes within the sandwich complexes and/or competitivebinding complexes of each localized sample oxidize the substratemolecules present in the aqueous media used to transport said substratemolecules. In embodiments in which sandwich complexes are present,oxidation occurs at an electrochemical cell formed by an electrochemicalsensor and the volume of fluid substantially over it and electrons flowfrom the working electrode of the electrochemical sensor to the volumesubstantially above the sensor in a quantity proportional to a quantityof target analyte present within the localized sample (e.g., magneticparticle bound complexes and/or surface bound complexes). In embodimentsin which competitive binding complexes are present, oxidation occurs atan electrochemical cell formed by an electrochemical sensor and thevolume of fluid substantially over the sensor and electrons flow fromworking electrode of the electrochemical sensor in a quantity inverselyproportional to a quantity of target analyte present within thelocalized sample.

At block 822, the reader's processor receives from the reader's electricconnector a first signal detected at the positive control workingelectrode within the analysis channel in the cartridge. In variousembodiments, the signal is a voltage or current or resistivity signal.At least a portion of the signal may be caused by the oxidation of thesubstrate bound to surface bound antibodies at the positive controlworking electrode. At block 824, the reader's processor receives fromthe reader's electric connector a second signal detected at the workingelectrode within the analysis channel in the cartridge. In variousembodiments, the signal is a voltage or current or resistivity signal.At least a portion of the signal is caused by the oxidation of thesubstrate over the working electrode, e.g., substrates magneticallybound to magnetic particles magnetically held over the workingelectrode. At block 826, the reader's processor receives from thereader's electric connector a third signal detected by a negativecontrol working electrode within the analysis channel in the cartridge.At block 828, the reader's processor processes and analyzes the signalfrom the working electrode (and, optionally, the signal from thepositive control working electrode and/or the signal from the negativecontrol working electrode) to identify the presence and/or quantity ofone or more target analytes. The reader's processor may determinewhether the parameter(s), e.g., current, voltage, of the first signalare within a predetermined range stored in the reader's memory, e.g., ina lookup table. Alternatively, the predetermined range may be stored inthe memory of the cartridge, stored in the memory for the device runningthe software application, or stored in a server in the system's network.The first signal may be used for error detection and diagnostic offaulty cartridges. The third signal may be indicative of noise presentwithin the system. The reader's processor may subtract or apply anotheralgorithm to remove the third signal from the second signal to accountfor and/or eliminate noise that may be present within the system.Alternatively, the third signal may be used for error detection anddiagnostic of faulty cartridges. Optionally, as shown at block 830, thereader may transmit signals indicative of a test result to a mobilecomputing device for further processing, storage, transmission to aserver, and/or display of results to a user.

The Charger

FIG. 25A illustrates a perspective view of an exemplary charger and FIG.25B is an exploded view illustrating the internal components of thecharger of FIG. 25A. Charger 500 is an optional feature that may be usedto charge reader device 400. Charger 500 may include alignment magnets501, inductive coil 502, and batteries 503. Charger 500 may include oneor more alignment magnets 501 configured to align with one or morealignment magnets in the reader device to facilitate efficient energytransfer between inductive coil 502 and the inductive coil within thereader. Illustratively, four magnets are used. Magnets 501 may be keyedto align with a corresponding keyed magnets in the reader. Charger 500may be plugged into a conventional socket, e.g., via a cord or a cordwith an AC to DC power converter, for charging components within charger500 such as batteries 503 to permit charging of reader 400.

The Reactants and the Reactions

Various devices, systems, kits, and methods disclosed herein areintended to isolate, tag, and detect one or more target analytes withina sample taken from a specimen. Chemical reactions may be employed toenable such detection. Chemical reactions may take place in a reservoirsuch as the sample preparation reservoir described above. For example,the sample preparation reservoir may hold a fluid such as water, salinesolution, water/saline solution mixed with one or more of magneticparticles, affinity molecules, connection molecules, signaling agents,competitor binding molecules, competitor molecules, labels, and/orsignaling agents. One or more reagent balls also may hold one or more ofmagnetic particles, affinity molecules, connection molecules, signalingagents, competitor binding molecules, competitor molecules, labels,and/or signaling agents outside of the fluid in the sample preparationreservoir. A reaction may begin when a sample potentially having one ormore target analytes (and, optionally, one or more reagent balls) is(are) mixed with fluid in the sample preparation reservoir, e.g., byintroducing a sample collected on a distal portion of a samplecollection device into the sample preparation reservoir when the samplecollection device is fully inserted into an input tunnel of a cartridge.Exemplary chemical reactions are discussed below and depicted in FIGS.26A-28H.

Referring to FIGS. 26A and 26C, target analyte 910 a, 910 b is added toa solution of sample preparation reagents, e.g., in the samplepreparation reservoir. Target analyte 910 a, 910 b may be any moleculesuch as a nucleic acid, protein, small molecule, or heavy metal or inthe case of a biologic or large molecule, the target analyte is afragment thereof associated with a particular condition or possiblecontamination or presence of a particular cell type, e.g., to detect atarget biomarker present on the surface of cell. Non-limiting examplesinclude specific pathogens, e.g. bacteria, viruses, parasites; toxins;hormones; immune regulatory molecules; or detectable fragments thereof.

Target analyte 910 a, 910 b may be detected using the systems andmethods described herein to for studying a condition such as molecularlevels indicative of inflammation, influenza, testosterone, fertility,and/or Vitamin D. The sample preparation reagents may include magneticmicrobeads or nanoparticles 920 a, 920 b (referred to herein as“magnetic particles”). Magnetic particles 920 a, 920 b are magneticallyresponsive such that they will be attracted to a magnetic field emittedfrom one or more magnetic generators. In this manner, magnetic particles920 a, 920 b released from the sample preparation reservoir traveldownstream in the analysis channel until they are localized over the oneor more working electrodes of the sensor within the analysis channel ofthe cartridge responsive to magnetic fields generated by one or moremagnetic generators of the reader. Each magnetic particle 920 a, 920 bmay have affinity molecule 930 a, 930 b bound to its surface. Themagnetic particles may be different sizes and may have a diameterbetween 50 nanometers to 5000 nanometers, between 100 nanometers to 4000nanometers, between 100 nanometers to 3000 nanometers, between 100nanometers to 2000 nanometers, between 100 nanometers to 1000nanometers, between 500 nanometers to 4000 nanometers, between 1000nanometers to 4000 nanometers, or between 1500 nanometers to 3000nanometers.

The affinity molecule may be any suitable molecule or moiety that canbind to or capture a target molecule. Non-limiting examples of affinitymolecules include antibodies (including single chain, multi-chainantibodies, diabodies, humanized antibodies, etc.), antibody fragmentswith affinity, ligands, polypeptide or protein molecules and moietieswith binding affinity for substrates, nucleic acid molecules (e.g.,aptamers), other molecules with binding affinity, and the like. Theaffinity molecules include chemically modified naturally occurringmolecules. Affinity molecules for a particular target analyte may beselected according to generally understood methods. For example, methodsof generating antibodies and fragments thereof are well known in theliterature and are exemplified by Antibodies: A Laboratory Manual (1988)Eds. Harlow and Lane, Cold Spring Harbor Laboratories Press, and U.S.Pat. Nos. 4,381,292, 4,451,570, and 4,618,577. Further, affinitymolecules are commercially available for specific target analytes. Alisting of such sources may be found in Linscott's Directory ofImmunological and Biological Reagents.

As used herein the terms “hybridize” and “hybridization” intend thespecific interaction between two entities, such as an antigen andantibody or two complementary nucleic acids, such that specific binding(covalent or non-covalent) can occur.

FIGS. 26A and 26B depict antibody 930 a and FIGS. 26C and 26D depictnucleic acid probe 930 b, although any suitable affinity molecule couldbe used, including a nucleic acid aptamer or other binding protein ormolecule. The sample preparation reagents also may include detectoragent 940 a, 940 b, such as, for example, an antibody 960 a conjugatedto signaling agent 950 a (FIG. 26A) or labeled nucleic acid probe 960 bbound to signaling agent 950 b (FIG. 26C). Detector agents 940 may eachinclude signaling agent 950, such as, for example, an oxidizing enzymeor other signaling enzyme, alkaline phosphatase (AP), methylene blue orother electrochemically responsive tag, or a fluorescent tag such asethidium bromide, fluorescein, green fluorescent protein, or otherfluorophore.

In embodiments that include detector agents 940, the various reagentslisted above may hybridize together to form sandwich complexes.Exemplary sandwich complexes 900 a, 900 b are illustrated in FIGS. 26Band 26D. Each sandwich complex may be formed of: (1) magnetic particle920 a, 920 b having surface-bound affinity molecule 930 a, 930 b, (2)target analyte 910 a, 910 b, and (3) detector agent 940 a, 940 b. Assuch, sandwich complex 900 a, 900 b held over the working electrode ofthe sensor within the analysis channel of the cartridge due to themagnetic attraction between magnetic particle 920 a, 920 b and themagnetic field generators of the reader will include target analyte 910a, 910 b and detector agent 940 a, 940 b. The exemplary sandwich complex900 a of FIG. 26B uses antibodies as affinity molecules, and the targetanalyte is a protein or small molecule of interest. The exemplarysandwich complex 900 b of FIG. 26D uses nucleic acid probes designed tocapture a particular sequence of nucleic acids. Also, competitormolecules may be used where the competitor molecules are each pre-boundto HRP, such as testosterone-HRP.

In various embodiments, signaling agent 950 a, 950 b is an oxidizingenzyme such as, for example, horseradish peroxidase (HRP) or soybeanperoxidase (SBP). In such embodiments, the enzyme induces an oxidationreaction to occur at an electrochemical cell when in the presence of aparticular chemical substrate, e.g., released from the substratereservoir, such as TMB and/or OPD which may be in a substrate solutionincluding acceptor molecules such as hydrogen peroxide. Thus, if theparticular substrate flows over, or otherwise encounters, the oxidizingenzyme bound to a target analyte and magnetic particle at anelectrochemical cell, an oxidation reaction occurs. In such embodiments,electrons are accordingly released from a working electrode of theelectrochemical cell to replenish electrons stripped from the substrateby the oxidizing enzyme in a quantity proportional to the amount oftarget analyte present. The release or flow of electrons results in acurrent, which is detectable by the sensor in the analysis channel(e.g., at the working electrode), for example, as a change in current ora change in voltage. Advantageously, signaling agents that are not boundto magnetic particles are not magnetically held over the workingelectrode and instead wash further downstream (e.g., caused by releaseof fluid from the sample preparation, wash, and/or substrate reservoirs)so as to not interfere with sensor readings. Accordingly, a signalindicative of the presence, absence, and/or quantity of one or moretarget analytes within a sample may be generated at the sensor in thecartridge and transmitted to the reader for further processing. Inaddition, or alternatively, surface bound target analytes with HRP mayalso produce signal that may be sensed by the sensor and transmitted tothe reader for further processing.

Referring now to FIGS. 27A and 27B, the sample preparation reagents mayinclude a population of magnetic particles 1020, each having affinitymolecule 1030 bound to its surface. Competitive binding agent 1040 and asample containing target analyte 1010 may be added to the samplepreparation reagents, e.g., by mixing the collected sample (and,optionally, one or more reagent balls) with fluid in the samplepreparation reservoir. Competitive binding agent 1040 may includepre-bound target analyte 1070, which comes pre-bound to signaling agent1050, for example, any of the signaling agents described above. Thepre-bound target analyte 1070 may be indirectly bound to the signalingagent 1050, for example, via an antibody, a nucleic acid probe, anucleic acid aptamer, or other affinity molecule 1060. Unbound targetanalyte 1010 from a sample and competitive binding agent 1040 maycompete with each other to bind to affinity molecules 1030 on magneticparticles 1020. The amount of competitive binding agent 1040 andsignaling agent 1050 that successfully binds to magnetic particles 1020is inversely proportional to the amount of target analyte 1010 presentin a sample. In embodiments where signaling agent 1050 of competitivebinding agent 1040 is an oxidizing enzyme, an oxidation reaction occursif a particular substrate (e.g., from the substrate reservoir) flowsover, or otherwise encounters, magnetic particles 1020 bound tocompetitive binding agents 1040 at the sensor within the analysischannel of the cartridge. Electrons are accordingly released from aworking electrode of the sensor to replenish electrons stripped from thesubstrate by the oxidizing enzyme in a quantity inversely proportionalto the amount of target analyte present in the sample. The release orflow of electrons results in a current, which is detectable by anelectrode coupled to electrical circuitry with a current-to-voltagetopology. Accordingly, a signal indicative of the presence, absence,and/or quantity of one or more target analytes within a sample may begenerated at the sensor in the cartridge and transmitted to the readerfor further processing.

Referring now to FIGS. 28A through 28H, a process for detecting thepresence, absence, and/or quantity of a target analyte(s) within asample in a cartridge is described. As shown in FIG. 28A, a samplehaving a plurality of sample target analytes 1110 may be collected withsample collection device 1100. Sample collection device 1100 may beconstructed in the manner described above with respect to any of thesample collection devices. Each sample target analyte 1110 may be boundto sample binding molecule 1115. Sample target analyte 1110 may be anytype of analyte described herein such as 25-hydroxy vitamin D3 or25-hydroxy vitamin D2 or 1α,25-dihydroxyvitamin D2 or1α,25-dihydroxyvitamin D. Sample binding molecule 1115 of the pluralityof sample binding molecules may be any type of binding molecule such asthe naturally occurring vitamin D binding protein also known asgc-component (group-specific component).

One or more reagent balls may be provided for mixing with the collectedsample. The reagent ball(s) may be stored within the cartridge, e.g.,within a shuttle stored in the cartridge as described above. Referringto FIG. 28B, reagent ball 1116 may include a plurality of competitormolecules 1170, a plurality of labels 1175, and a plurality ofcompetitor binding molecules 1180. Each competitor molecule 1170 may bepre-bound to competitor binding molecule 1180 of the plurality ofcompetitor binding molecules. Each competitor molecule 1170 may possesslabel 1175 of the plurality of labels. Competitor molecule 1170 may beany type of competitor molecule such as a 25-hydroxy vitamin D2 or25-hydroxy vitamin D3 possessing a label. Label 1175 may be any type oflabel such as biotin, and competitor binding molecule 1180 may be anytype of competitor binding molecule such as vitamin D binding protein.Each label 1175 is configured to bind to signaling agent 1150, e.g., viaattachment or affinity molecule 1160. In addition, or alternatively,label 1175 acts as signaling agent 1150. Reagent ball 1117 may include aplurality of solid particles 1120, a plurality of affinity molecules1130, a plurality of detector agents 1140 which may each have signalingagent 1150 and other affinity molecule 1160, and a plurality ofde-binding agents 1190. Reagent balls 1116, 1117 may be constructed inthe manner, and used in a cartridge as, described above with respect toany reagent ball described herein including reagent balls 375, 375′,375″, 375′″.

The plurality of solid particles 1120 may comprise magneticallyresponsive material such as the magnetic particles, as described herein,or non-magnetically responsive material such as gold nanoparticles.Preferably, each solid particle 1120 is bound to affinity molecule 1130,as described herein. Affinity molecule 1130 may be any affinity moleculedescribed herein and preferably has an affinity to bind to sample targetanalyte 1110 and/or competitor molecule 1170. Detector agent 1140 havingsignaling agent 1150 and affinity molecule 1160 may be similar to therespective agent/molecule described herein. For example, signaling agent1150 may be HRP and affinity molecule 1160 may be streptavidin. Eachde-binding agent 1190 of the plurality of depending agents is configuredto de-bind competitor molecule 1170 from competitor binding molecule1180 and, in some embodiments, to then bind to competitor bindingmolecule 1180, and/or to de-bind sample target analyte 1110 from samplebinding molecule 1115 and, in some embodiments, to then bind the samplebinding molecule 1115.

The molecules may be lyophilized in multiple reagent balls configured tobe used with a single cartridge or one reagent ball configured to beused with a single cartridge. The types of molecules may be distributedamongst the reagent balls in a desired manner, for example as shown inFIG. 28B, or randomly. In addition, certain types of molecules may bestored in the fluid within the sample preparation reservoir while othertypes are stored within a reagent ball(s).

As shown in FIG. 28C, all the molecules of reagent ball 1116 and reagentball 1117 of FIG. 28B may be lyophilized in a single reagent ball.Reagent ball 1118 may include a plurality of solid particles 1120′, aplurality of affinity molecules 1130′, a plurality of detector agents1140′ (having a plurality of signaling agents 1150′ and a plurality ofaffinity molecules 1160′), a plurality of competitor molecules 1170′, aplurality of labels 1175′, a plurality of competitor binding molecules1180′, and/or a plurality of de-binding agents 1190′. Reagent ball 1118may be constructed in the manner, and used in a cartridge as, describedabove with respect to any reagent ball described herein includingreagent balls 375, 375′, 375″, 375′″.

Referring now to FIG. 28D, a plurality of solid particles, a pluralityof affinity molecules, a plurality of detector agents (having aplurality of signaling agents and a plurality of affinity molecules), aplurality of competitor molecules, a plurality of labels, a plurality ofcompetitor binding molecules, and/or a plurality of de-binding agentsmay be mixed in fluid held in sample preparation reservoir 1119 with asample collected with sample collection device 1100. The sample may beintroduced into sample preparation reservoir 1119 while on the distalportion of sample collection device 1100 or after being released fromthe distal portion of sample collection device 1100, both of which aredescribed above. The sample may have a plurality of sample targetanalytes that may each be pre-bound to sample binding molecules. Theplurality of solid particles, plurality of affinity molecules, pluralityof detector agents (having the plurality of signaling agents and theplurality of affinity molecules), plurality of competitor molecules,plurality of labels, plurality of competitor binding molecules, and/orplurality of de-binding agents may be pre-stored in the fluid withinsample preparation reservoir 1119 or some or all of those molecules maybe introduced into the fluid from a reagent ball(s), such as reagentballs 1116, 1117, 1118. Sample preparation reservoir 1119 may beconstructed in the same manner as any sample preparation reservoirdescribed herein including sample preparation reservoirs 317, 317′,317″, 317′″.

Referring now to FIG. 28E, sample target analyte 1110 having samplebinding molecule 1115 pre-bound thereto may further mix within samplepreparation reservoir 1119. While not necessary, mixing within samplepreparation reservoir 1119 may be enhanced by actuation of a sonicatorelement disposed adjacent sample preparation reservoir 1119, asdescribed above.

As shown in FIG. 28F, de-binding agent 1190 may de-bind competitormolecule 1170 from pre-bound competitor binding molecule 1180 and thende-binding agent 1190 may bind to competitor binding molecule 1180leaving competitor molecule 1170 having label 1175 unbound. Anotherde-binding agent 1190 may de-bind sample target analyte 1110 from samplebinding molecule 1115 and then the other de-binding agent 1190 may bindto sample binding molecule 1190 leaving sample target analyte 1110unbound.

Referring to FIG. 28G, label 1175 of de-bound competitor molecule 1170may be configured to bind to signaling agent 1150 (e.g., via bondingwith affinity molecule 1160).

De-bound competitor molecule 1170 may be configured to bind to affinitymolecule 1130 of the plurality of affinity molecules which may bepre-bound to solid particle 1120 as shown in FIG. 28H. In this manner,solid particle 1120 may be indirectly bound to competitor molecule 1170indirectly bound to signaling agent 1150. A sandwich complex may beformed of solid particle 1120, affinity molecule 1130, competitormolecule 1170, label 1175, affinity molecule 1160, and/or signalingagent 1150. Illustratively, solid particle 1120 is bound to affinitymolecule 1130 which is bound to competitor molecule 1170 which is boundto label 1175 which is bound to affinity molecule 1160 which is bound tosignaling agent 1150.

Sample target analyte 1110 and competitor molecule 1170 may compete witheach other to bind to affinity molecules 1130 on solid particles 1120.The amount of competitor molecule 1170 and signaling agent 1150 thatsuccessfully binds to solid particles 1120 is inversely proportional tothe amount of unbound target analyte 1110 present in a sample. Inembodiments where signaling agent 1150 of competitive binding agent 1140is an oxidizing enzyme, an oxidation reaction occurs if a particularsubstrate (e.g., from the substrate reservoir) flows over, or otherwiseencounters, solid particles 1120 bound to competitor molecules 1140 atthe sensor within the analysis channel of the cartridge. Electrons areaccordingly released from a working electrode of the sensor to replenishelectrons stripped from the substrate by the oxidizing enzyme in aquantity inversely proportional to the amount of target analyte presentin the sample. For example, as shown in the graph of FIG. 29A comparingelectrochemical reading (microamps) versus concentration (ng/mL), thehigher the electrochemical reading, the lower the concentration ofsample target analytes. The release or flow of electrons results in acurrent, which is detectable by an electrode coupled to circuitry, forexample, as a change in current or a change in voltage. Accordingly, asignal indicative of the presence, absence, and/or quantity of one ormore target analytes within a sample may be generated at the sensor inthe cartridge and transmitted to the reader for further processing. FIG.29B shows a graph comparing electrochemical reading (microamps) versusconcentration (ng/mL) when competitor binding molecule is not pre-boundfor vitamin D quantification analysis. As FIG. 29B illustrates, theelectrochemical reading does not show a relationship with theconcentration of vitamin D.

As will be readily apparent to one skilled in the art, while one type ofmolecule may be shown as present in an illustrated reaction, forexample, one sample target analyte 1110 in FIGS. 28D through 28H, aplurality of the types of molecules may be present. In addition, all thetypes of molecules shown in FIGS. 28A through 28H need not be includedin a reaction. For example, referring to FIGS. 30A through 30H, thereactions are similar to FIGS. 28A through 28H except affinity molecule1130″ is not bound to a solid particle and the plurality of solidparticles are not needed.

Referring now to FIGS. 31A through 31H, a process for detecting thepresence, absence, and/or quantity of a target analyte(s) within asample in a cartridge is described. As shown in FIG. 31A, a samplehaving a plurality of sample target analytes 1210 may be collected withsample collection device 1200. Sample collection device 1200 may beconstructed in the manner described above with respect to any of thesample collection devices. Sample target analyte 1210 may be any type ofanalyte described herein.

One or more reagent balls may be provided for mixing with the collectedsample. The reagent ball(s) may be stored within the cartridge, e.g.,within a shuttle stored in the cartridge as described above. Referringto FIG. 31B, reagent ball 1215 may include a plurality of solidparticles 1120, a plurality of affinity molecules 1230, a plurality ofdetector agents 1240 which may each include signaling agent 1250 andaffinity molecule 1260, a plurality of control targets 1270, and/or aplurality of control detector agents 1275 which may each include controlsignaling agent 1285 and control affinity molecule 1280. Reagent ball1215 may be constructed in the manner, and used in a cartridge as,described above with respect to any reagent ball described hereinincluding reagent balls 375, 375′, 375″, 375′″.

The plurality of solid particles 1220 may comprise magneticallyresponsive material such as the magnetic particles, as described herein,or non-magnetically responsive material such as gold nanoparticles. Theplurality of solid particles 1220 may be similar to the magneticparticles described above. Preferably, each solid particle 1220 ispre-bound to affinity molecule 1230, as described herein. Affinitymolecule 1230 may be any affinity molecule described herein andpreferably has an affinity to bind to sample target analyte 1210.Detector agent 1240 having signaling agent 1250 and affinity molecule1260 may be similar to the respective agent/molecule described herein.For example, signaling agent 1250 may be HRP and affinity molecule 1260may be streptavidin. The plurality of control targets 1270 may beconfigured to bind to affinity molecules pre-bound to the surface of thesensor of the cartridge, for example, affinity molecules pre-bound tothe positive control working electrode of the sensor (e.g., see FIGS.7D, 7E, 7F) and/or the working electrode of the sensor (e.g., see FIG.7D). Control targets 1270 may be a protein. Control affinity molecule1280 may be any affinity molecule described herein and preferably has anaffinity to bind to control target 1270. Control signaling agent 1285may be similar to the signaling agents described herein, includingsignal agent 1250.

The molecules may be lyophilized in multiple reagent balls configured tobe used with a single cartridge or one reagent ball configured to beused with a single cartridge. The types of molecules may be distributedamongst the reagent balls in a desired manner or randomly. In addition,certain types of molecules may be stored in the fluid within the samplepreparation reservoir while other types are stored within a reagentball(s).

Methods encapsulate materials are known in the art but have not, to thebest of Applicants' knowledge, been utilized to encapsulate reactionreagents for use in a device, cartridge or system as described herein.As noted above, the reagent ball(s) may include one or more of magneticparticles, affinity molecules, connection molecules, signaling agents,competitor binding molecules, competitor molecules, labels, signalingagents, primers, nucleic acid probes, and/or polymerases, and otherenzymes or components as described in further detail herein and thecomponents, encapsulation material and dimensions may be the same ordifferent from each other.

Referring now to FIG. 31C, a plurality of solid particles, a pluralityof affinity molecules, a plurality of detector agents (having aplurality of signaling agents and a plurality of affinity molecules), aplurality of control targets, and/or a plurality of control detectoragents (having a plurality of control signaling agents and a pluralityof control affinity molecules) may be mixed in fluid held in samplepreparation reservoir 1290 with a sample collected with samplecollection device 1200. The sample may be introduced into samplepreparation reservoir 1290 while on the distal portion of samplecollection device 1200 or after being released from the distal portionof sample collection device 1200, both of which are described above. Thesample may have a plurality of sample target analytes. The plurality ofsolid particles, the plurality of affinity molecules, the plurality ofdetector agents (having the plurality of signaling agents and theplurality of affinity molecules), the plurality of control targets,and/or the plurality of control detector agents (having the plurality ofcontrol signaling agents and the plurality of control affinitymolecules) may be pre-stored in the fluid within sample preparationreservoir 1290 or some or all of those molecules may be introduced intothe fluid from a reagent ball(s), such as reagent ball 1215. Samplepreparation reservoir 1290 may be constructed in the same manner as anysample preparation reservoir described herein including samplepreparation reservoirs 317, 317′, 317″, 317′″.

Referring now to FIG. 31D, sample target analyte 1210 may further mixwithin sample preparation reservoir 1290. While not necessary, mixingwithin sample preparation reservoir 1290 may be enhanced by actuation ofa sonicator element disposed adjacent sample preparation reservoir 1290,as described above. Affinity molecule 1230 (pre-bound to solid particle1220) may bind with sample target analyte 1210 and detector agent 1240may bind to sample target analyte 1210. For example, affinity molecule1260 (pre-bound to signaling agent 1250) may bind to sample targetanalyte 1210. A sandwich complex may be formed of solid particle 1220,affinity molecule 1230, target analyte 1210, affinity molecule 1260,and/or signaling agent 1250. Illustratively, solid particle 1220 isbound to affinity molecule 1230 which is bound to target analyte 1210which is bound to affinity molecule 1260 which is bound to signalingagent 1250. Control detector agent 1275 may bind to control target 1270.For example, control affinity molecule 1280 (pre-bound to signalingagent 1285) may bind to control target 1270. A partial sandwich complexmay be formed of target control 1270, control affinity molecule 1280,and/or control signaling agent 1285. Illustratively, control target 1270is bound to control affinity molecule 1280 which is bound to controlsignaling agent 1285.

Referring now to FIG. 31E, a surface of a sensor for use in a cartridgedevice described herein is shown. Sensor surface 1292 may include aplurality of affinity molecules 1294 pre-bound to sensor surface 1292within the cartridge. Surface affinity molecule 1294 may be any affinitymolecule described herein and preferably has an affinity to bind tocontrol target 1270. Sensor surface 1292 is preferably positioned withinthe analysis channel of the cartridge for exposure to fluid releasedfrom the sample preparation reservoir and/or fluid released from thesubstrate reservoir. FIG. 31E shows sensor surface 1292 prior toexposure to fluid from the reservoir(s). Sensor surface 1292 may be usedon any of the sensors described above including sensors 338, 338′, 338″,338′″, 338″″. Sensor surface 1292 may be used for a working electrode ofthe sensor such as working electrode 340″ and/or may be used for apositive control working electrode of the sensor such as positivecontrol working electrodes 376, 376′, 376″.

Referring now to FIG. 31F, another surface of a sensor for use in acartridge device described herein is shown. Sensor surface 1296 may havea self-assembled monolayer such as thiolated ethylene glycol and/or adithiol such as hexaethylene glycol dithiol for added stability. Sensorsurface 1296 is preferably positioned within the analysis channel of thecartridge for exposure to fluid released from the sample preparationreservoir and/or fluid released from the substrate reservoir. FIG. 31Fshows sensor surface 1296 prior to exposure of fluid from thereservoir(s). Sensor surface 1296 may be used on any of the sensorsdescribed above including sensors 338, 338′, 338″, 338′″, 338″″. Sensorsurface 1296 may be used for a working electrode of the sensor such asworking electrodes 340, 340′, 340′″, 340″″. Sensor surface 1296 isconfigured to be exposed to magnetic fields from magnetic fieldgenerator 1298, e.g., when the cartridge is inserted in the reader. Forexample, magnetic field generator 1298 may be similar to first magneticgenerator 406 and second magnetic generator 407 of reader 400 describedabove.

Referring now to FIG. 31G, sensor surface 1292 is shown after exposureto reagents, e.g., from fluid flowing into the analysis channel from thesample preparation reservoir. As shown in FIG. 31G, partial sandwichcomplexes of control molecules may bind to surface affinity molecules1294 to complete the sandwich complexes. For example, surface affinitymolecule 1294 may bind to control target 1270 which is bound to controlaffinity molecule 1280 which is bound to control signaling agent 1285. Achemical reaction may occur when the sandwich complexes are exposed to asubstrate, e.g., from substrate reservoir, such that the sensor maydetect electrical signals resulting from chemical reactions over thesensor. For example, the mixed fluid from the sample preparationreservoir may be introduced into the analysis channel such that controlsignaling agents 1285 directly or indirectly bound to control targets1270 from the mixed fluid from the sample preparation reservoir localizeover sensor surface 1292 by binding with pre-bound surface affinitymolecules 1294. The chemical reactions may occur when fluid from thesubstrate reservoir reacts with particles from the mixed fluid from thesample preparation reservoir localized over the sensor. For example,substrate solution having a substrate may be introduced from thesubstrate reservoir and the sensor having sensor surface 1292 may detectelectrical signals resulting from the reactions between the substrate(e.g., TMB, OPD) and the signaling agents (e.g., HRP, SBP) localizedover the sensor. The reactions may cause electrons to be stripped fromthe substrate by the signaling agents (which electrons may be donated toacceptor molecules of the substrate solution) thereby generatingelectrical signals detectable by the sensor. If sensor surface 1292 ison a working electrode, such detected electrical signals may be used togenerate the signal indicative of the presence, absence, and/or quantityof one or more analytes within the sample. If sensor surface 1292 is ona positive control working electrode, such detected electrical signalsmay be used for error detection. For example, if a parameter(s), e.g.,voltage, current, of the detected electrical signals is not within apredetermined range(s), there may be an error and the test may berejected. If the parameter(s) is within the predetermined range,electrical signals detected by the working electrode of the sensor maybe used to generate the signal indicative of the presence, absence,and/or quantity of one or more analytes within the sample. The signalsfrom the positive control working electrode and/or the working electrodemay be transmitted to reader device 400, e.g., via respective electricalconnectors of cartridge device 300 and reader device 400.

Referring now to FIG. 31H, sensor surface 1296 is shown after exposureto reagents, e.g., from fluid flowing into the analysis channel from thesample preparation reservoir. It should be understood that sensorsurface 1296 and sensor surface 1292 may be exposed to the samplepreparation reservoir fluid at substantially the same time, e.g., whensensor surface 1296 corresponds to working electrode 340′″ or workingelectrode 340″″ and sensor surface 1292 corresponds to positive controlworking electrode 376′ of sensor 338′″ or positive control workingelectrode 376″ of sensor 338″″. Similarly, sensor surface 1296 andsensor surface 1292 may be exposed to the substrate reservoir fluid atsubstantially the same time, thereby causing reactions to occur betweenthe substrate and reagents (e.g., signaling agents) at substantially thesame time. A chemical reaction may occur when the sandwich complexes areexposed to a substrate, e.g., from substrate reservoir, such that thesensor may detect electrical signals resulting from chemical reactionsover the sensor. For example, the mixed fluid from the samplepreparation reservoir may be introduced into the analysis channel suchthat signaling agents directly or indirectly bound to target analytesfrom the mixed fluid from the sample preparation reservoir localize oversensor surface 1296 responsive to magnetic fields from magnetic fieldgenerator 1298 holding solid particles 1220 directly or indirectly boundto signaling agents 1250. The chemical reactions may occur when fluidfrom the substrate reservoir reacts with particles from the mixed fluidfrom the sample preparation reservoir localized over the sensor. Forexample, a substrate solution having a substrate may be introduced fromthe substrate reservoir and the sensor having sensor surface 1296 maydetect electrical signals resulting from the reactions between thesubstrate (e.g., TMB, OPD) and the signaling agents (e.g., HRP, SBP)localized over the sensor. The reactions may cause electrons to bestripped from the substrate by the signaling agents (which electrons maybe donated to acceptor molecules in the substrate solution) therebygenerating electrical signals detectable by the sensor. If sensorsurface 1296 is on a working electrode, such detected electrical signalsmay be used to generate the signal indicative of the presence, absence,and/or quantity of one or more analytes within the sample. The signalfrom the working electrode may be transmitted to reader device 400,e.g., via respective electrical connectors of cartridge device 300 andreader device 400.

As will be readily apparent to one skilled in the art, while one type ofmolecule may be shown as present in an illustrated reaction, forexample, one sample target analyte 1210 in FIGS. 31C and 31D, aplurality of the types of molecules may be present. In addition, all thetypes of molecules shown in FIGS. 31A through 31H need not be includedin a reaction.

Referring now to FIGS. 32A through 32I, a process for detecting thepresence, absence, and/or quantity of a target analyte(s) within asample in a cartridge is generally described. The process may involveamplification such as isothermal amplification as shown in general inFIG. 32D. As used herein, the term “isothermal amplification” refers toa method of amplifying a nucleic acid, e.g. DNA or RNA, wherein thetemperature remains constant. In one aspect, the reaction system is incontact with an outside source having a temperature differential but thetemperature change, if any, occurs slowly enough to allow the system tocontinuously adjust the temperature. Non-limiting exemplary ofisothermal amplification include rolling circle amplification (RCA),loop-mediated isothermal amplification (LAMP), strand displacementamplification (SDA), recombinase polymerase amplification (RPA),helicase dependent amplification (HDA), polymerase spiral reaction(PSR), and nicking enzyme amplification reaction (NEAR). These andfurther non-limiting exemplary methods are disclosed in Zhao et al.(2015) Chem. Rev. 115(22):12491-12545; and Dong et al. (2015) ScientificReports 5:12723; Yan et al. (2014) Mol. BioSyst. 10:970-1003.

Depending on the isothermal amplification method selected, the reagentball(s) may include one or more amplification enzymes, e.g., reversetranscriptases, endonucleases (e.g. a nicking endonuclease, restrictionendonuclease, flap endonuclease, or endonuclease III), polymerases (e.g.a strand displacing polymerase), method specific primers or probes (e.g.a padlock probe, linked probe, or LAMP primers), helicases,recombinases, and/or single-stranded DNA binding proteins. The requisitecombination of reagents required to carry out each method of isothermalamplification is appreciated by an ordinary skilled artisan.Non-limiting exemplary combinations of reagents required to perform thevarious methods of isothermal amplification are described in ChinesePatent Application No. 104232622; U.S. Pat. Nos. 5,223,414; 6,410,278;5,455,166; 5,470,723; 5,714,320; 6,235,502; and 7,282,328.

As used herein, the term “dNTPs” intend the nucleotides that are thebuilding blocks for DNA. They principally include (d)ATP, (d)GTP,(d)CTP, (d)TTP and (d)UTP and are necessary for nucleic acidamplification reactions.

RT specific primers intends a primer, typically unlabelled, that isdesigned to hybridize with an RNA molecule containing the target regionand allowing for a reverse transcriptase to polymerize a cDNA strandfrom the 3′ end of the primer. The cDNA strand will is then used as atemplate for isothermal amplification. The RT specific primer may havedesign characteristics that differ from the primers that are optimizedfor amplification of DNA within the isothermal amplification reactionthat proceeds subsequently to the reverse transcription of the targetRNA. Applicant has determined that use of RT specific primers willincrease amplification efficiency.

As shown in FIG. 32A, a sample having a plurality of sample targetanalytes 1310 and/or 1312 may be collected with sample collection device1300. Sample collection device 1300 may be constructed in the mannerdescribed above with respect to any of the sample collection devices.Sample target analyte 1310 may be any type of analyte described hereinand illustratively is RNA. Sample target analyte 1312 may be any type ofanalyte described herein and illustratively is DNA. It should beunderstood that sample target analyte 1310 and/or 1312 may contain anupstream and downstream sequence region of the target region, but isunderstood to at least contain the target region to be amplified anddetected. Sample target analyte 1310 and/or 1312 may be contained with acell, microbe, or virus or could be cell-free. The sample preparationreservoir and/or the reagent ball(s) may include lysis agents to freethe target analyte from the cell, microbe, or virus.

One or more reagent balls may be provided for mixing with the collectedsample. The reagent ball(s) may be stored within the cartridge, e.g.,within a shuttle stored in the cartridge as described above. Referringto FIG. 32B, reagent ball 1315 may include a plurality of solidparticles 1320, a plurality of affinity molecules 1322, a plurality ofdetector agents 1324 which may each include signaling agent 1326 andaffinity molecule 1328, a plurality of control targets 1330, a pluralityof control detector agents 1332 which may each include control signalingagent 1334 and control affinity molecule 1336, a plurality of solidparticles 1338 pre-conjugated to primers 1340 (e.g., via affinitymolecule 1342 pre-bound to capture element 1344 pre-bound to spacer 1346pre-bound to primer 1340 or through a direct covalent bond), a pluralityof detector agents 1348 which may each include signaling agent 1350 andaffinity molecule 1352, a plurality of internal control reverse primers1354 which may each be pre-bound to signaling agent 1356, e.g., eachoptionally via spacer 1358, a plurality of reverse primers 1360 whichmay each be pre-bound to label 1362, e.g., via spacer 1364, a pluralityof internal control forward primers 1366 which may each be pre-bound tocapture element 1368, e.g., via spacer 1370, a plurality of polymerase1372, a plurality of reverse transcriptases (RT) 1374, a plurality ofdNTPs 1376, a plurality of RT specific primers 1378, a plurality of RNAtemplate controls 1380, a plurality of DNA template controls 1382, aplurality of reverse primers 1384 which may each be pre-bound tosignaling agent 1386, e.g., via spacer 1388, a plurality of forwardprimers 1390 which may each be pre-bound to capture element 1392, e.g.,via spacer 1394, and/or a plurality of internal control reverse primers1396 which may each be pre-bound to internal control label 1394, e.g.,via internal control spacer 1400. Reagent ball 1315 may be constructedin the manner, and used in a cartridge as, described above with respectto any reagent ball described herein including reagent balls 375, 375′,375″, 375′″.

The plurality of solid particles 1320 may comprise magneticallyresponsive material such as the magnetic particles, as described herein,or non-magnetically responsive material such as gold nanoparticles. Theplurality of solid particles 1320 may be similar to the magneticparticles described above. Preferably, each solid particle 1320 ispre-bound to affinity molecule 1322, as described herein. Affinitymolecule 1322 may be any affinity molecule described herein andpreferably has an affinity to bind to a sample target analyte(s).Detector agent 1324 having signaling agent 1326 and affinity molecule1328 may be similar to the respective agent/molecule described herein.For example, signaling agent 1326 may be HRP and affinity molecule 1328may be streptavidin. The plurality of control targets 1330 may beconfigured to bind to affinity molecules pre-bound to the surface of thesensor of the cartridge, for example, affinity molecules pre-bound tothe positive control working electrode of the sensor (e.g., see FIGS.7D, 7E, 7F) and/or the working electrode of the sensor (e.g., see FIG.7D). Control targets 1330 may be a protein. Control affinity molecule1336 may be any affinity molecule described herein and preferably has anaffinity to bind to control target 1330. Control signaling agent 1334may be similar to the signaling agents described herein, includingsignal agent 1326. The plurality of solid particles 1338 may comprisemagnetically responsive material such as the magnetic particles, asdescribed herein, or non-magnetically responsive material such as goldnanoparticles. The plurality of solid particles 1338 may be similar tothe magnetic particles described above. Preferably, each solid particle1338 is pre-bound to affinity molecule 1342, as described herein.Affinity molecule 1342 may be any affinity molecule described herein andpreferably has an affinity to bind to capture element 1344. For example,affinity molecule 1342 may be pre-bound to capture element 1344. Captureelement 1344 contains a spacer 1346 and the primer 1340. Detector agent1348 having signaling agent 1350 and affinity molecule 1352 may besimilar to the respective agent/molecule described herein. For example,signaling agent 1350 may be HRP and affinity molecule 1352 may bestreptavidin. Affinity molecule 1352 may have an affinity to bind to alabel such as label 1362 and/or internal control label 1398. Internalcontrol reverse primers 1354 may be configured to bind to affinitymolecules pre-bound to the surface of the sensor of the cartridge, forexample, affinity molecules pre-bound to the positive control workingelectrode of the sensor (e.g., see FIGS. 7D, 7E, 7F) and/or the workingelectrode of the sensor (e.g., see FIG. 7D). Internal control reverseprimers 1354 may bind to affinity molecules pre-bound to the surface ofthe sensor via capture element 1368 bound to internal control forwardprimer 1366 (e.g., via spacer 1370). Signaling agent 1356 may be anysignaling agent described herein such as HRP. Reverse primers 1360 maybe configured to bind to the target analyte(s) and to bind directly orindirectly to a solid particle to localize at the surface of the sensorof the cartridge, for example, via magnetic fields at the workingelectrode of the sensor. Label 1362 may be any type of label such asbiotin and is configured to bind with an affinity molecule, e.g.,affinity molecule 1352 bound to signaling agent 1350. Internal controlforward primers 1366 may be configured to bind to affinity moleculespre-bound to the surface of the sensor of the cartridge, for example,affinity molecules pre-bound to the positive control working electrodeof the sensor (e.g., see FIGS. 7D, 7E, 7F) and/or the working electrodeof the sensor (e.g., see FIG. 7D) via, for example, capture element1368. The plurality of polymerase 1372, the plurality of reversetranscriptases (RT) 1374, the plurality of dNTPs 1376, the plurality ofRT specific primers 1378, the plurality of RNA template controls 1380,and/or the plurality of DNA template controls 1382 may be used toamplify the nucleic acid, e.g., RNA and/or DNA, via, for example,isothermal amplification. The plurality of RNA template controls 1380,and/or the plurality of DNA template controls 1382 may be contained witha cell, microbe, or virus or could be free floating. The samplepreparation reservoir and/or the reagent ball(s) may include lysisagents to free the template control(s) from the cell, microbe, or virus.Reverse primers 1384 may be configured to bind to a target analyte(s)and to bind directly or indirectly to a solid particle to localize atthe surface of the sensor of the cartridge, for example, via magneticfields at the working electrode of the sensor. Signaling agent 1386 maybe any signaling agent described herein such as HRP. Forward primers1390 may be configured to bind to a target analyte and to bind directlyor indirectly to a solid particle to localize at the surface of thesensor of the cartridge, for example, via magnetic fields at the workingelectrode of the sensor via, for example, capture element 1392. Internalcontrol reverse primers 1396 may be configured to bind to affinitymolecules pre-bound to the surface of the sensor of the cartridge, forexample, affinity molecules pre-bound to the positive control workingelectrode of the sensor (e.g., see FIGS. 7D, 7E, 7F) and/or the workingelectrode of the sensor (e.g., see FIG. 7D). Internal control reverseprimers 1396 may bind to affinity molecules pre-bound to the surface ofthe sensor via capture element 1368 bound to internal control forwardprimer 1366 (e.g., via spacer 1370). Label 1398 may be any type of labelsuch as biotin and may be configured to bind with an affinity molecule,e.g., affinity molecule 1352 bound to signaling agent 1350.

The molecules may be lyophilized in multiple reagent balls configured tobe used with a single cartridge or one reagent ball configured to beused with a single cartridge. The types of molecules may be distributedamongst the reagent balls in a desired manner or randomly. In addition,certain types of molecules may be stored in the fluid within the samplepreparation reservoir while other types are stored within a reagentball(s).

Referring now to FIG. 32C, any combination of the types of particlesshown in reagent ball 1315 may be mixed in fluid held in samplepreparation reservoir 1290 with a sample collected with samplecollection device 1200. The sample may be introduced into samplepreparation reservoir 1410 while on the distal portion of samplecollection device 1300 or after being released from the distal portionof sample collection device 1300, both of which are described above. Thesample may have a plurality of sample target analytes. The types ofparticles shown in reagent ball 1315 may be pre-stored in the fluidwithin sample preparation reservoir 1410 or some or all of thosemolecules may be introduced into the fluid from a reagent ball(s), suchas reagent ball 1315. Sample preparation reservoir 1410 may beconstructed in the same manner as any sample preparation reservoirdescribed herein including sample preparation reservoirs 317, 317′,317″, 317′″.

Sample target analyte 1310 and/or 1312 may further mix within samplepreparation reservoir 1410. While not necessary, mixing within samplepreparation reservoir 1410 may be enhanced by actuation of a sonicatorelement disposed adjacent sample preparation reservoir 1410, asdescribed above. The mixing within sample preparation reservoir 1410 mayamplify nucleic acids, e.g., DNA and/or RNA, within the mixture of fluidwithin sample preparation reservoir which includes the sample andoptionally reagent ball(s). The sample target analyte may be a targetnucleic acid, e.g., a RNA for the detection and/or diagnosis of aninfluenza or HIV infection, and DNA for DNA viruses or bacteria or RNAfor the detection of 16 s rRNA for bacteria. The amplification proceedsby action of a polymerase beginning at the 3′ end of a bound primer(forward or reverse) wherein the polymerase moves along the templatestrands comprising the target nucleic acid and incorporates nucleotides(dNTP) to synthesize a complementary strand. Since the primer may have alabelled end, these labelled ends are incorporated into the resultingamplicon. In the case of a capture element labelled end, this allow theamplicon to bind to an affinity molecule bound to a solid particle or toa pre-bound surface affinity molecule on a sensor surface while thesignaling agent on the other end of the amplicon can be conjugateddirectly or indirectly to the signaling agent. As used herein,“conjugated” intends a covalent or non-covalently bond, e.g., abiotin-Strepavidin binding complex. And these binding events can happensimultaneously with the amplification. In a further aspect, a separateamplification reaction is simultaneously occurring and lysis of viralparticles, bacteria, other microbes, and cells may be occurring freeingthe cellular contents, e.g., nucleic acid. In addition to the labeledprimers, unlabeled primers designed to amplify at least the same targetregion but perhaps some sequence flanking the target sequence may beprovided to facilitate the amplification reaction simultaneously withthe labelled primers. The presence of unlabeled primers can makeamplification more efficient since labelled primers may be moresterically hindered as they bind to other elements e.g. solid particlesor signaling agents.

This polymerization of the complement strand in the amplification can befacilitated by the presence of single stranded binding proteins known tothose of ordinary skill in the art such as but not limited to SSB fromE. coli. Strand displacing polymerases can be used to allow for thepolymerization of the complement strand to occur without thermallydenaturing the double strand prior to synthesis of complementarystrands. Since the primer may have a labelled end, these labelled endsare incorporated into the resulting amplicon. In the case of a captureelement labelled end, this allow the amplicon to bind to an affinitymolecule bound to a solid particle or to a pre-bound surface affinitymolecule on a sensor surface while the signaling agent on the other endof the amplicon can be conjugated directly or indirectly to thesignaling agent. As used herein, “conjugated” intends a covalent ornon-covalently bond, e.g., a biotin-Strepavidin binding complex. Andthese binding events can happen simultaneously with the amplification.

The amplification may be an isothermal reaction such as isothermalamplification. FIG. 32D shows an exemplary process for isothermalamplification of nucleic acids. The amplification of nucleic acidswithin sample preparation reservoir 1410 generates amplicons as shown inFIG. 32E. Actuation of the sonication element to mix the contents of thesample preparation reservoir may promote the amplification. Theamplicons may form sandwich complexes which are labeled ampliconcomplexes 1422 comprising a capture label and a signaling label, orlabeled amplicon complexes 1434. For example, a forward primer andreverse primer amplicon may be bound to a signaling agent and/or boundto a solid particle as a result of the mixing. Amplicon complexes 1422and 1434 may each include a solid particle configured to be magneticallyheld over a working electrode in a sensor such that a signaling agent ofthe complex can react with a substrate from a substrate solution. Theamplicons may form partial sandwich complexes which may be unlabeledpartial amplicon complexes 1420 or labeled partial amplicon complexes1426. For example, an internal control forward primer and an internalcontrol reverse primer amplicon may be bound to a signaling agent as aresult of the mixing. Partial amplicon complexes 1420 and 1426 may beconfigured to bind to pre-bound surface affinity molecules over anelectrode (e.g., working electrode and/or positive control workingelectrode) of a sensor.

Pre-conjugated primers (either covalently or through a linkage such asbiotin-streptavidin) may be used wherein either the forward or thereverse is conjugated to a particle and the other side (forward ifreverse was conjugated to particle and reverse if forward was conjugatedto particle) is conjugated to a signaling agent either directly orindirectly and there may also be a population of unlabelled andunconjugated primers that facilitates the reaction to occur moreefficiently.

Referring now to FIG. 32F, a surface of a sensor for use in a cartridgedevice described herein is shown. Sensor surface 1412 may include aplurality of affinity molecules 1414 pre-bound to sensor surface 1412within the cartridge. Surface affinity molecule 1414 may be any affinitymolecule described herein and preferably has an affinity to bind to apartial amplicon complex. For example, surface affinity molecule 1414may have an affinity to bind to an internal control capture elementbound to an internal control forward primer and an internal controlreverse primer amplicon bound to a signaling agent. The internal controlreverse primer amplicon may be bound to a signaling agent, e.g., via aspacer, or may be bound to a label, e.g., via a spacer, which is boundto an affinity molecule bound to a signaling agent. Sensor surface 1412is preferably positioned within the analysis channel of the cartridgefor exposure to fluid released from the sample preparation reservoirand/or fluid released from the substrate reservoir. FIG. 32F showssensor surface 1412 prior to exposure to fluid from the reservoir(s).Sensor surface 1412 may be used on any of the sensors described aboveincluding sensors 338, 338′, 338″, 338′″, 338″″. Sensor surface 1412 maybe used for a working electrode of the sensor such as working electrode340″ and/or may be used for a positive control working electrode of thesensor such as positive control working electrodes 376, 376′, 376″.

Referring now to FIG. 32G, another surface of a sensor for use in acartridge device described herein is shown. Sensor surface 1416 may havea self-assembled monolayer such as thiolated ethylene glycol and/or adithiol such as hexaethylene glycol dithiol for added stability. Sensorsurface 1416 is preferably positioned within the analysis channel of thecartridge for exposure to fluid released from the sample preparationreservoir and/or fluid released from the substrate reservoir. FIG. 32Gshows sensor surface 1416 prior to exposure of fluid from thereservoir(s). Sensor surface 1416 may be used on any of the sensorsdescribed above including sensors 338, 338′, 338″, 338′″, 338″″. Sensorsurface 1416 may be used for a working electrode of the sensor such asworking electrodes 340, 340′, 340′″, 340″″. Sensor surface 1416 isconfigured to be exposed to magnetic fields from magnetic fieldgenerator 1418, e.g., when the cartridge is inserted in the reader. Forexample, magnetic field generator 1418 may be similar to first magneticgenerator 406 and second magnetic generator 407 of reader 400 describedabove.

Referring now to FIG. 32H, sensor surface 1414 is shown after exposureto reagents, e.g., from fluid flowing into the analysis channel from thesample preparation reservoir. As shown in FIG. 32H, partial ampliconcomplexes 1420 of control amplicon molecules may bind to surfaceaffinity molecules 1414 to complete the amplicon complexes. For example,surface affinity molecule 1414 may bind to an internal control captureelement bound to an internal control forward primer amplicon and aninternal control reverse primer amplicon bound to a signaling agent asshown in FIG. 32H. A chemical reaction may occur when the ampliconcomplexes are exposed to a substrate, e.g., from substrate reservoir,such that the sensor may detect electrical signals resulting fromchemical reactions over the sensor. For example, the mixed fluid fromthe sample preparation reservoir may be introduced into the analysischannel such that the signaling agents directly or indirectly bound tothe amplicon primers from the mixed fluid from the sample preparationreservoir localize over sensor surface 1412 by binding with pre-boundsurface affinity molecules 1414, e.g., via capture elements. Thechemical reactions may occur when fluid from the substrate reservoirreacts with particles from the mixed fluid from the sample preparationreservoir localized over the sensor. For example, a substrate solutionhaving a substrate may be introduced from the substrate reservoir andthe sensor having sensor surface 1412 may detect electrical signalsresulting from the reactions between the substrate (e.g., TMB, OPD) andthe signaling agents (e.g., HRP, SBP) localized over the sensor. Thereactions may cause electrons to be stripped from the substrate by thesignaling agents (which electrons may be donated to acceptor moleculesfrom the substrate solution) thereby generating electrical signalsdetectable by the sensor. If sensor surface 1412 is on a workingelectrode, such detected electrical signals may be used to generate thesignal indicative of the presence, absence, and/or quantity of one ormore analytes within the sample. If sensor surface 1412 is on a positivecontrol working electrode, such detected electrical signals may be usedfor error detection. For example, if a parameter(s), e.g., voltage,current, of the detected electrical signals is not within apredetermined range(s), there may be an error and the test may berejected. If the parameter(s) is within the predetermined range,electrical signals detected by the working electrode of the sensor maybe used to generate the signal indicative of the presence, absence,and/or quantity of one or more analytes within the sample. The signalsfrom the positive control working electrode and/or the working electrodemay be transmitted to reader device 400, e.g., via respective electricalconnectors of cartridge device 300 and reader device 400.

Referring now to FIG. 32I, sensor surface 1416 is shown after exposureto reagents, e.g., from fluid flowing into the analysis channel from thesample preparation reservoir. It should be understood that sensorsurface 1416 and sensor surface 1412 or sensor surface 1424 may beexposed to the sample preparation reservoir fluid at substantially thesame time, e.g., when sensor surface 1416 corresponds to workingelectrode 340′″ or working electrode 340″″ and sensor surface 1412corresponds to positive control working electrode 376′ of sensor 338′″or positive control working electrode 376″ of sensor 338″″. Similarly,sensor surface 1416 and sensor surface 1412 or sensor surface 1424 maybe exposed to the substrate reservoir fluid at substantially the sametime, thereby causing reactions to occur between the substrate andreagents (e.g., signaling agents) at substantially the same time.

As shown in FIG. 32I, amplicon complexes 1422 of target ampliconmolecules may localize over sensor surface 1416. Target ampliconcomplexes 1422 may be formed of a solid particle bound to targetamplicon primers bound to a signal agent. For example, the solidparticle may be bound to an affinity molecule bound to a capture elementbound to a target forward primer amplicon and a target reverse primeramplicon bound to the signaling agent as shown in FIG. 32I. A chemicalreaction may occur when the target amplicon complexes are exposed to asubstrate, e.g., from substrate reservoir, such that the sensor maydetect electrical signals resulting from chemical reactions over thesensor. For example, the mixed fluid from the sample preparationreservoir may be introduced into the analysis channel such thatsignaling agents directly or indirectly bound to target amplicons fromthe mixed fluid from the sample preparation reservoir localize oversensor surface 1416 responsive to magnetic fields from magnetic fieldgenerator 1418 holding the solid particles directly or indirectly boundto the signaling agents. The chemical reactions may occur when fluidfrom the substrate reservoir reacts with particles from the mixed fluidfrom the sample preparation reservoir localized over the sensor. Forexample, a substrate solution having a substrate may be introduced fromthe substrate reservoir and the sensor having sensor surface 1416 maydetect electrical signals resulting from the reactions between thesubstrate (e.g., TMB, OPD) and the signaling agents (e.g., HRP, SBP)localized over the sensor. The reactions may cause electrons to bestripped from the substrate by the signaling agents (which electrons maybe donated to acceptor molecules from the substrate solution) therebygenerating electrical signals detectable by the sensor. If sensorsurface 1416 is on a working electrode, such detected electrical signalsmay be used to generate the signal indicative of the presence, absence,and/or quantity of one or more analytes within the sample. The signalfrom the working electrode may be transmitted to reader device 400,e.g., via respective electrical connectors of cartridge device 300 andreader device 400.

Referring now to FIG. 32J, sensor surface 1424 is similar to sensorsurface 1412 of FIG. 32H and the control amplicon complexes are similarto the control amplicon complexes of FIG. 32H except control ampliconcomplexes 1426 include a label bound to the internal control reverseprimer amplicon which is bound to an affinity molecule bound to asignaling agent. For example, surface affinity molecule 1428 may bind toan internal control capture element bound to an internal control forwardprimer amplicon and an internal control reverse primer amplicon bound toa label bound to an affinity molecule bound to a signaling agent asshown in FIG. 32J. The reaction and signal processing may occur asdescribed above with respect to FIG. 32H.

Referring now to FIG. 32K, sensor surface 1430 is similar to sensorsurface 1416 and magnetic field generator 1432 is similar to magneticfield generator 1418 of FIG. 32I and the target amplicon complexes aresimilar to the target amplicon complexes of FIG. 32I except targetamplicon complexes 1434 include a label bound to the target reverseprimer amplicon which is bound to an affinity molecule bound to asignaling agent. For example, a solid particle may be bound to anaffinity molecule bound to a capture element bound to a target forwardprimer amplicon and a target reverse primer amplicon bound to a labelbound to an affinity molecule bound to a signaling agent as shown inFIG. 32K. The reaction and signal processing may occur as describedabove with respect to FIG. 32I.

As will be readily apparent to one skilled in the art, while one type ofmolecule may be shown as present in an illustrated reaction, forexample, one solid particle 1320 in FIG. 32B, a plurality of the typesof molecules may be present. In addition, all the types of moleculesshown in FIGS. 32A through 32K need not be included in a reaction.

The sample reagents may include only one population of magneticparticles and one population of detector agents or competitive bindingagents. Such embodiments may be tailored for detection of a singletarget analyte of interest.

In other embodiments, multiple populations of magnetic particles anddetector agents and/or competitive binding agents and/or multiplepopulations of distinct affinity molecules are provided, each populationconstructed to have its own affinity. For example, each population ofmagnetic particles has a unique affinity molecule bound to its surface,and each population of magnetic particles is thereby designed to bindwith a different target analyte. Similarly, each population of detectoragents includes a unique affinity molecule and is thereby designed tobind with a different target analyte. A multiplexing scheme may be usedsuch that a first size of magnetic particles is more magneticallyresponsive to a first working electrode, and a second size of magneticparticles is more magnetically responsive to a second working electrode,etc. (third, fourth, fifth, etc. sizes and working electrodes may beused). Alternatively or additionally, different surface binding schemesmay be used for different working electrodes such that a first set ofaffinity molecules directed to a first target analyte population isimmobilized at the surface of a first working electrode and a second setof affinity molecules directed to a second target analyte population isimmobilized at the surface of a second working electrode of theelectrochemical cell, etc. (third, fourth, fifth, etc. affinities andworking electrodes may be used). In this manner, the respective targetanalyte population binds to the respective affinity molecules (andsignaling agent) and binds to the surface bound affinity molecules onthe surface of the respective working electrode. Accordingly, multiplesets of affinity molecules and addressable working electrodes may beused to execute a multiplexing scheme. One or more populations ofmagnetic particles having unique magnetic properties and/or uniqueaffinity molecules bound thereto, as described above, may be multiplexedwith one or more different surface binding schemes. For example, theprocess of FIGS. 31A-31H may occur at substantially the same time as theprocess of FIGS. 32A-32K within the same cartridge using the same ordifferent sample preparation reservoirs and the same or differentsensors. In embodiments employing the competitive binding approach, eachpopulation of competitive binding agents may include a differentpre-bound target analyte and is thereby designed to compete with adifferent target analyte. Such embodiments allow for the detection of aplurality of target analytes, including detection of multiple foodbornepathogens, multiple contaminants, and multiple ailments.

Those skilled in the art will appreciate that the possibilities forforming the magnetic particle-bound complexes are numerous and all suchpossibilities are contemplated herein. For example, the samplepreparation reagents may include a biotin-labelled antibody, which bindsto a portion of the target analyte. In some embodiments, antibodiesand/or nucleic acids present among the sample preparation reagents maybe pre-biotinylated such that a streptavidin conjugated signaling enzymecan bind with the biotinylated detector to form a complex. One suchstreptavidin conjugated signaling enzyme is HRP. The tagging combinationis not limited to biotin-streptavidin. Any suitable tagging scheme willwork. In another example, multiple HRP enzymes are conjugated togetherinto a molecule commonly known as a Poly-HRP molecule in order toenhance the signal generating capability of the resultant sandwichcomplex.

In addition to the components that form the magnetic particle-boundcomplexes, the sample preparation reagents of various embodiments mayinclude one or more of: (a) agents that facilitate formation of themagnetic particle-bound complexes, such as salts; (b) agents thatfacilitate access and specificity to target analytes, such as detergentsand enzymes for lysis of bacteria or viruses or cutting of largemolecules or nucleotides; (c) blocker proteins to decrease nonspecificbinding; and (d) stabilizers such as, for example, trehalose, which canimprove the shelf life of the sample preparation reagents.

For the sample preparation reagents, salts may be necessary to enhancethe likelihood of binding. For example, phosphate buffered saline (PBS)may be the fluid held in the sample preparation reservoir. Any saltwhich does not interfere with electrochemical detection may be providedwithin the reagents.

Blocker proteins, such as the well-known Bovine Serum Albumin, casein,fibrinogen, or other blocker protein may be provided to help stabilizethe antibodies, enzymes, and/or other proteins present among the samplepreparation reagents. Such blocker proteins may also help preventnon-specific binding of signaling enzymes to the magnetic particles andto the walls of the systems and devices described elsewhere herein.

Additionally, for embodiments that require lysis to access the moleculesor nucleic acids of interest, detergents may be employed. In variousembodiments, nonionic detergents, rather than ionic detergents, areprovided to prevent denaturation of the signaling enzyme and/orantibodies. Detergents may enhance lysis of bacteria, but are alsouseful for gently lysing various viruses, such as the influenza virus.Such lysing may be desirable to improve access to target analytes suchas nucleoproteins internal to a virus. Additionally, the samplepreparation reagents may include enzymes that enhance lysis and reduceviscosity during lysis; such reagents may be necessary for thepreparation of some samples, for example, samples containing bacteriasuch as E. coli. The enzymes that enhance and facilitate lysis mayinclude lysozymes and DNAses that chop up released genomic DNA withoutdisrupting nucleic acid probes on the surface of the magnetic particles.

Enzymes such as RNAses or DNAses, which selectively chop largernucleotide sequences into smaller sequences, may be useful forgenerating smaller fragments having favorable binding kinetics. Suchenzymes may be present in the sample preparation reagents. Othercomponents also may be included within the sample preparation reagents.For example, a stabilizer agent such as trehalose, may be present; suchstabilizer agents help protect proteins from oxidation and therebyincrease the shelf-life of the reagents, especially at room temperature.

Referring now to FIG. 33, detection system 100 is shown in use wheresample collection device 200 is inserted in cartridge device 300 whichis inserted in reader device 400. Reader device 400 may transmit signalsindicative of the presence, absence, and/or quantity of one or moretarget analytes to a mobile device running software-based detectioninterface system 600 such that a user may review and interact with theanalyzed results on the presence, absence, and/or quantity of one ormore target analytes. Such results may be transmitted to server 1500 vianetwork 1510. Software-based detection interface system 600 may bedownloaded onto the mobile device. Software-based detection interfacesystem 600 may be a dedicated application or “app” and may be downloadedfrom an online store such as iTunes™ (Apple, Inc., Cupertino, Calif.),the App Store (Apple, Inc.), Google™ Play (Google, Inc., Mountain View,Calif.), the Android™ Marketplace (Google, Inc.), Windows™ Phone Store(Microsoft Corp., Redmond, Wash.), or BlackBerry™ World (BlackBerry,Waterloo, Ontario, Canada). Preferably, software-based detectioninterface system 600 need only be downloaded once—although updates maybe downloaded.

Sample collection device 200 may be disposable and configured forone-time use. It may come within removable sterile packaging. Onceinserted into the input tunnel of cartridge device 300, samplecollection device 200 may be locked into a permanent fixed engagementand cannot be used again. Cartridge device 300 also may be disposableand configured for one-time use. Once sample collection device 200 locksinto place within the input tunnel of cartridge 300, cartridge 300cannot be used again. Cartridge 300, may, however, be removed from thereader 400. Cartridge 300 and reader 400 may be configured to beseparably coupled, and cartridge 300 may be inserted and removed fromthe dock of reader 400 at least before and after implementation of adetection protocol. Reader 400 may include a locking mechanism fortemporarily locking cartridge 300 into place, and limiting removal,during the duration of a detection test cycle. Reader 400 of variousembodiments is reusable.

Reader 400, and the entire detection system 100, may be configured fornon-clinical, consumer-directed use. Accordingly, system 100 is easy touse and generates results quickly. Results of a target analyte detectionprotocol may be generated in 30 minutes or less from the time a samplefrom sample collection device 200 is inserted into the system'scartridge 300. Results may be generated in less than 20 minutes, lessthan 10 minutes, and/or less than 5 minutes. Additionally, theconsumer-directed system may be small for an unobtrusive presence withina home, school, office, or other place of employment. Cartridge 300,sample collection device 200, and reader 400 together may beapproximately the size of a smartphone or other mobile computing device.System 100 may be sized and configured to be portable. In suchembodiments, in addition to a compact, hand-held design, all fluidswithin the sample are properly sealed and separated such that no leakingor premature oxidation reactions will occur due to jostling of thesystem components while on the go.

To promote use by lay people in non-clinical settings, system 100 may bedesigned to be “dummy proof” by including a self-activating and self-rundetection protocol. For example, FIG. 33 depicts an example in whichcartridge 300 has been placed into the dock of reader 400 and samplecollection device 200 has been inserted into the input tunnel ofcartridge 300. In the depicted embodiment, loading cartridge 300 intoreader 400 may establish an electrical connection between the pins ofcartridge 300 and reader 400, thereby completing a circuit within reader400, which automatically activates reader 400. Upon being activated,reader 400 may determine if sample collection device 200 is properlyinserted in cartridge 300, e.g., upon receipt of an electrical signalindicating that the contact switch in cartridge 300 has been activated.Upon detection, reader 400 may initiate a detection protocolautomatically without any further human intervention. The automatedstart ensures that mixing of reagents and sample within the samplepreparation reservoir occurs consistently at a fixed time followinginsertion of sample collection device 200, leading to consistent testresults. Alternatively, the testing protocol may initiate when a userpresses a “go”, “run”, “start”, or other similar button or icon onreader 400 or computing device 601 running software 600.

In various embodiments, computing device 601 may be included within thesystem: to provide for more computing power and/or more memory; toprovide a wireless transceiver for pulling data from, and transmittingdata to, a remote server; and/or to provide a display screen and userinterface. Computing device 600 is not needed within every embodiment.

One skilled in the art will appreciate that the embodiment in FIG. 33 isillustrative in nature only and various components may be added,deleted, or substituted and various different hierarchies and modes ofcommunication between the devices may be employed. Communication network1510 through which some or all of the various devices communicate withone another. The network may be a local area network (LAN) or a widearea network (WAN). Network 1510 may be a wireless communicationnetwork, such as, for example, a mobile WiMAX network, LTE network,Wi-Fi network, or other wireless network. The communication betweencomputing device 601 having a user interface software 600 and server1500 may occur over the internet via a wired network, such as a DSLcable connection.

Communication between reader 400 and computing device 601 may occur,wirelessly, for example, using Bluetooth®, Wi-Fi, near-fieldcommunications, or other radiofrequency technology. Alternatively,transmission of signals between reader 400 and computing device 601 mayoccur over a cord, cable, or other wired or direct connection. Invarious embodiments, computing device 601 or other device having a userinterface software 600 includes a software application for a front-end,graphical user interface for presenting test results to a user.

Reader 400 may be configured to control the tests and processes neededto detect and/or quantify one or more target analytes within a sample.To do so, a significant amount of information may be stored within thememory of reader 400. Alternatively, some or all of the information maybe stored within computing device 601 and/or server 1500 and accessibleby reader 400 via the communication network 1510. Such information mayinclude, for example, a database of cartridge keys, which identifieseach cartridge type by the signal generated by the cartridge's uniqueidentifier stored in the cartridge's memory. The information also mayinclude test protocols associated with each type of cartridge. The testprotocols may specify details such as how long to mix sample preparationreagents through sonication, the frequency of the sonication, when toheat the various heat-sensitive valves, etc. The information may alsoinclude correlation tables for each cartridge type, which correlatedetected sensor signals to the absence, presence, and/or a specificquantity of a target analyte. Additionally, the information stored byreader 400 and/or server 1500 may include one or more past results.Reader 400 may store test results at least until reader 400 comes intocommunication with a remote computing device; at such time, the resultsmay be transmitted to the remote computing device (e.g., computingdevice 601, server 1500) for display and/or long-term storage.

Server 1500 also may store user profiles, which may include biographicalinformation entered into the system by a user through computing device601 having user interface software 600. A log of test results for eachuser may also be stored by server 1500 and accessible for viewing by theuser through transmission of such data to computing device 601 with userinterface software 600.

DEFINITIONS

Unless otherwise defined, each technical or scientific term used hereinhas the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. In accordance with the claimsthat follow and the disclosure provided herein, the following terms aredefined with the following meanings, unless explicitly stated otherwise.

The term “about” or “approximately,” when used before a numericaldesignation or range (e.g., pressure or dimensions), indicatesapproximations which may vary by (+) or (−) 5%, 1% or 0.1%.

As used in the specification and claims, the singular form “a”, “an” and“the” include both singular and plural references unless the contextclearly dictates otherwise. For example, the term “a molecule” mayinclude, and is contemplated to include, a plurality of molecules. Attimes, the claims and disclosure may include terms such as “aplurality,” “one or more,” or “at least one;” however, the absence ofsuch terms is not intended to mean, and should not be interpreted tomean, that a plurality is not conceived.

As used in the specification and claims, “at least one of” meansincluding, but not limited to, one or more of any combination of thefollowing. For example, “at least one of A, B, and C” or “at least oneof A, B, or C” means including, but not limited to, A(s) or B(s) or C(s)or A(s) and B(s) or A(s) and C(s) or B(s) and C(s) or A(s) and B(s) andC(s); none of which excludes other elements such as D(s), E(s), etc.

As used herein, the term “comprising” or “comprises” is intended to meanthat the devices, systems, and methods include the recited elements, andmay additionally include any other elements. “Consisting essentially of”shall mean that the devices, systems, and methods include the recitedelements and exclude other elements of essential significance to thecombination for the stated purpose. Thus, a device or method consistingessentially of the elements as defined herein would not exclude othermaterials or steps that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanthat the devices, systems, and methods include the recited elements andexclude anything more than a trivial or inconsequential element or step.Embodiments defined by each of these transitional terms are within thescope of this disclosure.

Although the foregoing has included detailed descriptions of someembodiments by way of illustration and example, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof these embodiments that numerous changes and modifications may be madewithout departing from the spirit or scope of the appended claims.

What is claimed is:
 1. A sample analysis cartridge comprising a housing:an input tunnel that extends from an aperture in the housing, the inputtunnel configured to permit insertion of a sample collection devicehaving a distal portion adapted to be exposed to a sample; a reservoirconfigured to hold a fluid, the reservoir further configured to receivethe sample collected by the sample collection device; a collet disposedin the input tunnel between the reservoir and the aperture, the colletcomprising a collet body having a collet proximal end and a colletdistal end, a deflector portion, and a lumen extending between thecollet proximal end and the collet distal end, the lumen sized toreceive the distal portion of the sample collection device therein; ashuttle at least partially disposed within the collet in a firstposition, the shuttle comprising a shuttle body having a shuttleproximal end and a shuttle distal end and defining a sample compartmentbetween the shuttle proximal end and the shuttle distal end, the shuttleconfigured to receive the sample collected by the sample collectiondevice in the sample compartment through an opening at the shuttleproximal end, the shuttle further configured to move within the inputtunnel from the first position to a second position responsive to forceapplied on the shuttle from the sample collection device duringinsertion of the sample collection device in the input tunnel; and acontact switch disposed adjacent the deflector portion of the collet,wherein the deflector portion is configured to deflect to activate thecontact switch responsive to force applied on the deflector portion bythe sample collection device during insertion of the sample collectiondevice in the input tunnel.
 2. The sample analysis cartridge of claim 1,wherein the shuttle is configured to move within the input tunnel fromthe first position to the second position after the collet is decoupledfrom the shuttle such that the shuttle is at least partially disposedwithin the reservoir in the second position and the sample is exposed tothe fluid in the reservoir.
 3. The sample analysis cartridge of claim 1,wherein the shuttle further defines a reagent ball compartment betweenthe shuttle proximal end and the shuttle distal end configured to housea reagent ball comprising reagents.
 4. The sample analysis cartridge ofclaim 3, wherein the reagent ball is disposed in the shuttle to not beexposed to the fluid in the reservoir in the first position and to beexposed to the fluid in the reservoir in the second position.
 5. Thesample analysis cartridge of claim 1, wherein the shuttle proximal endis configured to be disposed within the lumen of the collet in the firstposition.
 6. The sample analysis cartridge of claim 5, wherein theshuttle distal end forms at least a part of a wall of the reservoir inthe first position.
 7. The sample analysis cartridge of claim 1, whereinthe collet comprises one or more locking arms configured to couple thecollet to the shuttle in the first position.
 8. The sample analysiscartridge of claim 7, wherein the one or more locking arms areconfigured to be deflected to decouple the one or more locking arms fromthe shuttle responsive to force applied on the one or more locking armsby the sample collection device during insertion of the samplecollection device in the input tunnel.
 9. The sample analysis cartridgeof claim 1, further comprising a sensor configured to be exposed to thefluid mixed with the sample, the sensor further configured to generate asignal indicative of at least one of a presence, absence, or quantity ofthe one or more analytes within the sample.
 10. The sample analysiscartridge of claim 1, further comprising a sealing material configuredto fluidicly seal the fluid within the reservoir and a seal piercerdisposed partially within the input tunnel, the seal piercer configuredto be contacted by the sample collection device within the input tunneland to move, responsive to force applied by the sample collectiondevice, to pierce the sealing material to vent the fluid in thereservoir.
 11. The sample analysis cartridge of claim 10, wherein thecollet comprises a slot and a portion of the seal piercer extendsthrough the slot into the input tunnel to permit contact between theseal piercer and the sample collection device.
 12. The sample analysiscartridge of claim 1, wherein the deflector portion of the colletcomprises an arm configured to deflect downward to activate the contactswitch.
 13. The sample analysis cartridge of claim 1, wherein thecontact switch is positioned such that activation of the contact switchindicates full insertion of the sample collection device in the inputtunnel.
 14. The sample analysis cartridge of claim 1, wherein the colletfurther comprises one or more protrusions configured to couple to theshuttle proximal end in the first position.
 15. The sample analysiscartridge of claim 14, wherein the one or more protrusions each compriselead-in angles configured to guide the distal portion of the samplecollection device into the opening at the shuttle proximal end.
 16. Asample analysis cartridge comprising a housing: an input tunnel thatextends from an aperture in the housing, the input tunnel configured topermit insertion of a sample collection device having a distal portionadapted to be exposed to a sample; a reservoir configured to hold afluid, the reservoir further configured to receive the sample collectedby the sample collection device; a shuttle disposed in the input tunnelbetween the reservoir and the aperture in a first position, the shuttlecomprising a shuttle body having a shuttle proximal end and a shuttledistal end and defining a sample compartment between the shuttleproximal end and the shuttle distal end, the shuttle configured toreceive the sample collected by the sample collection device in thesample compartment through an opening at the shuttle proximal end, theshuttle further configured to move within the input tunnel from thefirst position to a second position responsive to force applied on theshuttle from the sample collection device during insertion of the samplecollection device in the input tunnel; and a collet disposed in theinput tunnel and coupled to the shuttle in the first position such thatthe shuttle is at least partially disposed within the collet in thefirst position, the collet comprising a collet body having a colletproximal end and a collet distal end and a lumen extending between thecollet proximal end and the collet distal end, the lumen sized toreceive the distal portion of the sample collection device therein, thecollet configured to decouple from the shuttle during insertion of thesample collection device in the input tunnel, wherein the shuttle isconfigured to move within the input tunnel from the first position tothe second position after the collet is decoupled from the shuttle suchthat the shuttle is at least partially disposed within the reservoir inthe second position and the sample is exposed to the fluid in thereservoir.
 17. The sample analysis cartridge of claim 16, wherein theshuttle proximal end is configured to be disposed within the lumen ofthe collet in the first position.
 18. The sample analysis cartridge ofclaim 16, wherein the shuttle distal end forms a wall of the reservoirin the first position.
 19. The sample analysis cartridge of claim 16,wherein the collet comprises one or more locking arms configured tocouple the collet to the shuttle in the first position.
 20. The sampleanalysis cartridge of claim 19, wherein the one or more locking arms areconfigured to be deflected to decouple the one or more locking arms fromthe shuttle responsive to force applied on the one or more locking armsby the sample collection device during insertion of the samplecollection device in the input tunnel.
 21. The sample analysis cartridgeof claim 16, further comprising a sealing material configured tofluidicly seal the fluid within the reservoir and a seal piercerdisposed partially within the input tunnel, the seal piercer configuredto be contacted by the sample collection device within the input tunneland to move, responsive to force applied by the sample collectiondevice, to pierce the sealing material to vent the fluid in thereservoir.
 22. The sample analysis cartridge of claim 21, wherein thecollet comprises a slot and a portion of the seal piercer extendsthrough the slot into the input tunnel to permit contact between theseal piercer and the sample collection device.
 23. The sample analysiscartridge of claim 16, further comprising a sensor configured to beexposed to the fluid mixed with the sample, the sensor furtherconfigured to generate a signal indicative of at least one of apresence, absence, or quantity of the one or more analytes within thesample.
 24. The sample analysis cartridge of claim 16, furthercomprising a contact switch, wherein the collet comprises a deflectorportion disposed adjacent the contact switch, the deflector portionconfigured to deflect to activate the contact switch responsive to forceapplied on the deflector portion by the sample collection device duringinsertion of the sample collection device in the input tunnel.
 25. Thesample analysis cartridge of claim 24, wherein the deflector portion ofthe collet comprises an arm configured to deflect downward to activatethe contact switch.
 26. The sample analysis cartridge of claim 24,wherein the contact switch is positioned such that activation of thecontact switch indicates full insertion of the sample collection devicein the input tunnel.
 27. The sample analysis cartridge of claim 16,wherein the collet further comprises one or more protrusions configuredto couple to the shuttle proximal end in the first position, the one ormore protrusions each comprising lead-in angles configured to guide thedistal portion of the sample collection device into the opening at theshuttle proximal end.
 28. The sample analysis cartridge of claim 16,wherein the shuttle further defines a reagent ball compartment betweenthe shuttle proximal end and the shuttle distal end configured to housea reagent ball comprising reagents, and wherein the reagent ball isdisposed in the shuttle to not be exposed to the fluid in the reservoirin the first position and to be exposed to the fluid in the reservoir inthe second position.